Lipid a and other carbohydrate ligand analogs

ABSTRACT

The core structure of pentaerythritol has been used as a replacement for one or both sugars in lipid A, leading to the generation of a series of lipid A analogs. These lipid A analogs may further differ from lipid A with respect to, e.g., the number, nature and location of negatively charged groups, and the number, nature and location of the lipid chains. The lipid A analogs may be lipid A agonists useful as immunostimulatory agents, or lipid A antagonists useful in the treatment of septic shock. In a like manner, a residue of pentaerythritylamine may be used as a replacement for an amino sugar residue in a carbohydrate ligand having a biological activity of interest, generating a series of ligand analogs. These are useful, e.g., as haptens, inhibitors of bacterial-host cell adhesion, etc.

This application is a continuation of U.S. patent application Ser. No.10/513,556, PCT filed May 9, 2003 and published as U.S. PatentApplication No. 2006/0040891, which is a national stage entry ofInternational Application No. PCT/US03/14633, filed May 9, 2003 andpublished as Pub. No. WO/2003/94850, and claims the benefit of priorityof U.S. Provisional Application Ser. No. 60/378,645, filed May 9, 2002,all of which are hereby incorporated by reference in their entirety.

CROSS-REFERENCE TO RELATED APPLICATIONS

Biomira (Jiang, et al.), PCT/US00/31281, filed Nov. 15, 2000 (our docketJIANG3A-PCT) relates to the design and synthesis of some new Lipid-Aanalogs. The analogs were monophosphorylated, and contained either (1)at least one novel and unnatural lipid (such as lipids I or II) ofcompounds 33 and 102 (its FIG. 3), or (2) an unnatural combination oflipids. The latter refers to those Lipid-A analogs that carry lipids ofuniform chain length. Its Compounds 54 and 86 (its FIG. 4) fall intothis category. Its Compound 70 (its FIG. 19) is similar, but it alsocontains an n-propyl group at 3-O-position and is an example of Lipid-Aanalog that incorporates a short unnatural alkyl group with an unnaturalether linkage. By using a synthetic lipopeptide antigen,(FIG. 34), amodified 25-amino-acid sequence that is derived from tumor-associatedMUC1 mucin, the applicants were able to evaluate the adjuvant propertiesof certain synthetic Lipid-A analogs disclosed in this invention. Basedon the data of T-cell blastogenesis and IFN-γ level obtained throughpreliminary in vivo/in vitro studies, it was demonstrated that syntheticLipid-A structures 48, 54, 70, 86, 102 and 104 are as effective, asadjuvants, as the Lipid-A preparations of bacterial origin.

Koganty, et al., U.S. Prov. Appl. No. 60/387,437, filed Jun. 11, 2002(Atty Docket: Koganty5) relates to combinatorial peptide andglycopeptide libraries utilizing a pentaerythritol core.

Biomira (Koganty et al.), PCT/US03/10750, filed Apr. 9, 2003 (AttyDocket: Koganty4A-PCT) teaches that a glycolipopeptide comprising atleast one disease-associated epitope, and characterized by at least onelipidated interior amino acid or by the presence of a MUC1 epitope, maybe used in a vaccine, preferably in conjunction with a liposome.

Biomira (Longenecker, et al.), PCT/US95/04540, filed Apr. 12, 1995 (ourdocket LONGENECKER5—PCT), discloses that a conjugate of a primaryepitope and an immunomodulatory peptide, or a mixture of a primaryantigen and an immunomodulatory peptide, may be used to elicit an immuneresponse which is CMI-specific.

Biomira (Jiang et al.), PCT/CA03/00135, filed Feb. 4, 2003, relates tothe use of covalently lipidated oligonucleotides comprising the CpGdinucleotide unit, or an analogue thereof, as immunostimulatory agents.It discloses that a Pet structure can be used to link together suchunits.

The above-noted related applications are hereby incorporated byreference in their entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to lipid A analogs characterized by thereplacement of a sugar unit by a derivative of pentaerythritol. It alsorelates to analogs of carbohydrate ligands, including lipid A,characterized by the replacement of an amino sugar unit by a derivativeof pentaerythritylamine.

2. Description of the Background Art

Pentaerythritol. Pentaerythritol (Pet) and di-pentaerythritol (di-Pet)are common polyols and they are widely used in oil industry to producelubricants and other macromolecules. A derivative,tetrakis-[13-(2′-deoxythymidin-3′-O-yl)-6,9-diaza-2-oxa-5,10,13-trioxotridecyl)-methane(dT₄-PE-PLC) has been used as a liquid phase carrier for large-scaleoligonucleotide synthesis in solution (Wörl, R. et al, 1999, compound6). In addition, Pet derivatives, semifluorinated pentaerythritoltetrabenzoates, have been employed to design liquid crystallinestructures (Cheng, X. H. et al, 2000) and pentaerythritol lipidderivatives (e.g., dimristoyl-trimethylglycine pentaerythritol) havebeen used in the preparation of cationic liposomes for the delivery ofnucleic acids into mammalian cells (Nantz, M. H. et al, 2001). Atriamine derivative of pentaerythritol has been used as a startingmaterial in the preparation of chelating agents (Dunn, et al., 1990).

The four-directional core (the “Pet” unit) of pentaerythritol has beenemployed successfully as a coupling agent, for example, in the synthesisof multifunctional dendrimers (Armspach, D. et al, 1996 and Kuzdzal, S.A. et al, 1994), and as a molecular scaffold for combinatorial chemistry(Farcy, N. et al, 2001). Furthermore, Ranganathan et al used the Petunit as a core to design a spiro-self-assembling cyclic peptide forconstructing twin nanotubes (Ranganathan, D. et al, 2001).

It is particularly interesting to note the use of the Pet unit to couplesugar units. Lindhorst, et al, Eur. J. Org. Chem., 2027-34 (2000) usedthe Pet unit as a framework for a cluster of four mannosides. Schmidt,et al., Eur. J. Org. Chem., 669-674 (2002) prepared similar structuresin which a lipid group (C16H33) was O-linked to one of the fourperipheral carbons, and one to three mannoside residues were O-linked,through an ethyleneoxy oligomeric spacer, to other of the peripheralcarbons. Those peripheral carbons which did not link to a lipid or to asugar-containing moiety were simply hydroxylated. Finally, Hanessian etal. 1996 used a pentaerythritol scaffold to present a cluster of two Tn(the monosaccharide GalNAc) or TF (the disaccharide D-Galβ (1->3)GalNAc)epitopes, each O-linked through a spacer to a peripheral carbon of thePet core. Of remaining two peripheral carbons, one was O-linked to—CH2CH2NHAc, and the other O-linked to either allyl (Hanessian 33) or1-octenyl (Hanessian 37). In none of these references was a peripheralcarbon of the Pet core N-linked to any moiety.

In the various applications mentioned above, the Pet unit serves as acore to carry other moieties. It may also be used to replace a sugarunit in an oligosaccharide. However, it has never before been used toreplace a sugar unit in the lipid A disaccharide. Nor has Pet-NH— beenused to replace an amino sugar in any carbohydrate ligand.

Toepfer et al disclosed sialyl-Lewis X and sialyl-Lewis A mimicscontaining one Pet unit (Toepfer et al. 1995; Toepfer et al. 2000) asnew ligands for selectin binding. Thus, in compound 4 of Toepfer et al.1995, two of the peripheral carbons of the Pet unit are hydroxylated,one is O-linked to a moiety comprising a single sugar unit, and the lastone is O-linked to a moiety comprising a disaccharide. It should benoted that in Toepfer's analogs, the Pet unit replaces a normal sugarunit, not an amino sugar as in applicants' carbohydrate ligand analogs.In addition, the only lipophilic groups contemplated by Toepfer et al.are groups customarily used as protecting groups in organic synthesis,such as those resulting in replacement of sugar hydroxyls with —O-All,—O-Tf, or —O-Bn.

Aguilera et al 1998 reported the testing of analogs of oligosaccharidesfor anti-mitotic activity. The original oligosacccharides were thetetrasaccharide α-D-GalNac-β-D-Gal-(1->4)-[α-L-Fuc-(1->3)]-β-D-GlcOMe,and a related sulfated trisaccharide (Aguilera compound 1), whichcontain a Lewis X-type structure. In the analogs of the trisaccharide(Aguilera compounds 13-16), one sugar was replaced with a Pet unit. Inthe analogs of the tetrasaccharide (17, 18), two of the sugar units werereplaced with Pet units. The analogs thus contained the disaccharide inwhich the a-fucosyl residue was linked to the C-3 position of theGlcNac. In all six analogs, one hydroxyl of the disaccharide moiety wasreplaced with —O(CH₂)₇CH₃, thus imparting a lipid function. In analogs14, 16 and 18, three of the four Pet unit peripheral carbons werehydroxylated (the remaining carbon being linked to a group comprisingthe disaccharide moiety). In Aguilera compounds 13, 15 and 17, twoperipheral Pet carbons were hydroxylated and the third was sulfated.However, these compounds were found to be inactive as antimitotic agentsin all of the cell types, thus discouraging further use of negativelycharged groups in analogs of this family.

Lipopolysaccharide (bacterial). Lipopolysaccharide (LPS) is a uniqueglycolipid found exclusively in the outer leaflet of the outer membraneof Gram-negative bacteria. Structurally, bacterial LPS molecule hasthree main regions: the O-antigen region, the core region and theLipid-A region (Stryer, 1981; Raetz, WO86/05687). The O-antigen regionis a strain-specific, polysaccharide moiety and determines the antigenicspecificity of the organism. The core region is an oligosaccharide chainand may play a role in maintaining the integrity of the outer membrane.The Lipid-A region is conserved and functions as a hydrophobic anchorholding lipopolysaccharide in place.

LPS is known to trigger many pathophysiological events in mammals,either when it is injected or when is accumulated due to Gram-negativebacterial infection. Before the discovery of Lipid-A component of LPSthe term “endotoxin” was generally used to describe the effects of theLPS. The endotoxin from Gram-negative bacteria is heat-stable, cellassociated, pyrogenic and potentially lethal. In addition to itsendotoxic activities, LPS also exhibits various biological activities,which include immuno adjuvant activity, B-lymphocyte mitogenesis,macrophage activation, interferon production, tumor regression, etc.While both the O-antigen and the core regions modulate the toxicactivity of the LPS, it is generally believed that the hydrophobicLipid-A moiety is responsible for these pathophysiological effects ofthe endotoxin (Rietschel, 1992: Takada, 1992).

Lipid A and Its Synthetic Analogs. Lipid A is the lipid anchor oflipopolysaccharide (LPS), the outer cell membrane component ofGram-negative bacteria. LPS is a strong activator of the innate immunityof the host following bacterial infection, and its lipid A moiety hasbeen shown to be responsible for the biological activities of LPS inmost in vitro and in vivo test systems. The structure-activityrelationships of lipid A and its analogs have been extensively studiedover the last two decades (Rietschel et al, 1996; Takada & Kotani,1989).

Lipid-A consists of a β-(1,6)-linked D-glucosamine disaccharidephosphorylated at 1-O- and 4′-O-positions. Hydroxylated andnon-hydroxylated fatty acids are linked to the hydroxyl and amino groupsof the disaccharide to confer hydrophobicity to the Lipid-A. FIG. 1 ofPCT/US00/31281 (Jiang3A-PCT) shows two examples of natural Lipid-Astructures, compound A (Imoto, 1985a, b) isolated from E. coli, andcompound B (Rietschel, 1984a, b; Seydel, 1981; Strain, 1985) isolatedfrom Salmonella strains.

A large number of synthetic lipid A analogs have been prepared. Forexample, Lien et al. 2001 describe the agonist ER-112022, in which thedisaccharide backbone of lipid A is replaced with —CH₂CH₂—NHCO—(CH₂)₄—CONH—CH₂CH₂—. The two phosphate groups link this substitutebackbone to the lipid chains. Christ et al. 1995 prepared the lipid Aantagonist E5531, derived by modification of the structure of theendotoxin-antagonistic Rhodobacter capsulatus lipid A, in which thenaturally occurring acyl linkages at the C-3 and C-3′ carbons werereplaced with ether linkages, and the C-6′ hydroxyl group was blocked.E5531 had advantages in stability and purity.

Takada and Kotani have conducted a thorough study of structuralrequirements of Lipid-A for endo-toxicity and other biologicalactivities (Takada & Kotani, 1989), comparing synthetic Lipid-A analogsprepared by various groups (Kotani, 1986a, b; Kiso, 1986: Fujishima,1987; Charon, 1985: Sato, 1995). They reported that for immunoadjuvantactivity, the structural requirements of Lipid-A do not appear to be asrigid as those required for endotoxic activity and IFN-α/β or TNF-alphainducing properties (Takada, 1989; Ribi, 1982). Removal of all fattyacids, however, abrogates all biological activities normally attributedto Lipid-A.

Ribi et al 1982 showed that the minimal structure required for toxicitywas a bisphosphorylated β-(1,6)-linked di-glucosamine core to which longchain fatty acids are attached. It appears that an optimal number oflipid chains, in the form of either hydroxy acyl or acyloxyacyl groups,are required on the disaccharide backbone in order to exert strongendotoxic and related biological activities of Lipid-A (Kotani, 1986a).

In addition, removal of either phosphate group results in significantloss of toxicity without a corresponding loss of adjuvant activity.Bioassays on monophosphoryl Lipid-A showed that, while it was 1000 timesless potent on a molar basis in eliciting toxic and pyrogenic responses,it was comparable to diphosphoryl Lipid-A (and endotoxin itself) inimmunostimulating activities (Werner, 1996). It is known that thediphosphoryl Lipid-A from E. coli and Salmonella strains are highlytoxic, but the monophosphoryl Lipid-A from E. coli has reduced toxicitywhile retaining the numerous biological activities that are normallyassociated with LPS (Werner, 1996; Takayama, 1984; Ulrich, 1995; Myerr,1990).

Recently, it was suggested that the agonistic and antagonist activity oflipid A were governed by the intrinsic conformation of lipid A, which inturn was defined mainly by the number of charges, the number anddistribution of acyl chains in in the molecule (Seydel et al 2000;Schromm et al, 2000).

Furthermore, lipid A has been suggested to be a ligand for Toll-likereceptor 4 (TLR4), a pattern-recognition receptor involved in themediation of immune responses to LPS/lipid A (Kutuzova et al, 2001).

There is a need for effective treatment for Lipid-A/LPS associateddisorders, and for a potent adjuvant without the associated toxicity.The high toxicity of unmodified Lipid-A from natural source discouragesits general use as a pharmaceutical.

Another major drawback with the naturally derived Lipid-A is inaccessing sufficient material with pharmaceutically acceptable purity,reproducible activity and stability. Naturally derived Lipid-A is amixture of several components of cell wall including those of Lipid-Awith varying number of lipid chains. Such heterogeneity in naturalLipid-A product is attributed to two sources: (1) biosyntheticvariability in the assembly of the Lipid-A moiety and (2) loss of fattyacids from Lipid-A backbone during processing and purification.Consequently, it is difficult to control the manufacturing process interms of reproducibility of composition of the mixture, which hassignificant bearing in biological activity and toxicity.

SUMMARY OF THE INVENTION Lipid A Analogs

One object of the present invention is the design, synthesis and use oflipid A analogs in which one or both of the sugar units of the lipid Adisaccharide is replaced with at least the carbon skeleton ofpentaerythritol (the Pet core). These lipid A analogs may becharacterized by additional differences from the natural product, e.g.,changes in the number, structure and location of the lipid chains,elimination of one phosphate group, replacement of one or both phosphategroups with a related group (e.g., a sulfate group), and changes in thespacing or linkage of the sugar units (or their replacements).

The present invention also includes lipid A analogs in which one of thesugar units of the lipid A disaccharide is replaced with at least thecarbon skeleton of pentaerythritol, and the other unit is omitted.

FIG. 3 shows a few examples of structural analogs of a lipid Adisaccharide obtained by employing one or two Pet units.

In some embodiments, the lipid A analog is a derivative ofpentaerythritamine (Pet-NH2), which is appropriate as lipid A comprisesglucosamine, an amino sugar.

Lipid A analogs having lipid A agonistic activity (immunostimulatoryactivity) are useful as immunotherapeutic agents for the treatment ofinfections and cancers. As vaccine adjuvants, they can be formulatedtogether with antigens to provide stronger immune responses to theadministered antigens and thereby improve vaccine efficacy. They canalso be used as stand-alone therapeutic agents (improving innateimmunity). Naturally, they may be used in combination with othertherapeutic agents for the treatment of targeted diseases.

Lipid A analogs having lipid A antagonistic activity may be used for thecontrol of LPS-mediated pathophysiological disorders. Due to theexaggerated response to LPS released from Gram-negative bacteria,bacterial infection can sometimes lead to a cascade ofpathophysiological events termed sepsis. Sepsis is deadly; it kills tensof thousands annually in the United States alone. Lipid A antagonistsmay bind to the LPS-binding receptor, Toll-like receptor 4 (TLR4), butsuch binding will not lead to the un-controlled release of inflammatorycytokines by the immune system. Therefore, these antagonists can beeffective therapeutics to treat LPS-mediated disorders, such asinflammation and septic shock symptoms.

In the present invention, we have designed a class of lipid A analogscomprising a Pet core, and synthesized several specific examples(compound 1-4, FIG. 5).

Preliminary biological data show that lipid A mimics 1 and 2, whichcontain one PetNH₂ unit replacing the reducing end glucosamine of lipidA disaccharide, exhibit strong immunostimulatory activities (FIGS. 14and 15). To further demonstrate the biological activity of thecontemplated analogs, each of synthetic lipid A analogs 1 and 2 wasincorporated into a liposomal formulation, together with a synthetictumor-associated lipopeptide antigen derived from tumor-associated MUC1glycoprotein. This vaccine formulation demonstrates obvious inhibitioneffect on tumor growth in mice (FIG. 16).

Thus, lipid A analogs, especially compounds 1 and 2, may be used asimmunostimulatory adjuvants in treating diseases, as disclosed in thisinvention. In a preferred embodiment, they are used in liposomalconstructs, which comprise totally synthetic immunostimulatoryadjuvant(s) and totally synthetic antigen(s), for immunologicallytreating various diseases, such as infectious diseases and cancers.

Two other lipid A analogs, 3 and 4, have been synthesized. These containderivatives of a di-pentaerythritol (di-Pet) and a di-pentaerythritamine(di-PetNH₂) unit, respectively, replacing the whole lipid A disaccharidebackbone. The biological properties of compound 3 and 4 have not beenevaluated.

Those analogs that possess lipid A antagonistic activity will be usefulin treating lipopolysaccharide (LPS)—endotoxin—related disorders, suchas septic shock.

Carbohydrate Hapten Analogs

Another object of the present invention is the design, synthesis and useof analogs of carbohydrate ligands. While a monosaccharide has severalchiral centers, the Pet unit does not possess any chiral center due toits high symmetry. It is because of this non-chiral property that Petcan mimic various monosaccharides of different stereochemistry.Similarly, when one arm of the Pet is substituted with an amino group,the resulting pentaerythritamine (PetNH₂) unit can mimic variousamino-substituted monosaccharides, including1-amino-1-deoxy-(glycosylamine), 2-amino-2-deoxy-(glycosamine),3-amino-3-deoxy-monosaccharide, etc. Therefore, structural mimics ofalmost all naturally occurring carbohydrate molecules can be produced byusing a combination of natural monosaccharides and Pet unit(s), or Petunit(s) alone.

While others have used the Pet unit to replace a sugar unit in acarbohydrate ligand, in every case the Pet unit employed was oneretaining all of the hydroxyloxygens, i.e., (Pet carbon core) (—O—)₄.The examples set forth below have demonstrated that derivatives (

Pet-NH—) of pentaerythritamine (PetNH₂) can readily mimic the reducingend glucosamine of lipid A disaccharide. Since lipid A is a carbohydrateligand comprising an amino sugar, it is tempting to assume that PetNH₂can be used to construct analogs of other carbohydrate ligands whichcomprise amino sugars, with

Pet-NH— replacing at least one of these amino sugars. For example

Pet-NH— can be used to replace N-acetyl-glucosamine andN-acetyl-galactosamine. Derivatives of the form (Pet carbon core)(—O—)₂—NH— are of particular interest.

FIG. 19 shows some PetNH₂-derived analogs of tumor-associatedcarbohydrate antigens, which are potentially useful for the developmentof immunotherapeutics to treat cancers. Similarly, FIG. 20 shows somePetNH₂-derived analogs of carbohydrate ligands involved in bacterialadhesion to host. Bacterial infection usually starts with thecolonization of bacteria onto the host cells, during which processcarbohydrate molecules are used as the binding ligands. Structuralanalogs of these carbohydrates are potential inhibitors for bacterialadhesion, and therefore can be effectively used as antibiotics toprevent bacterial infection. One advantage of such analogs over thenaturally occurring carbohydrates is that the analogs are more resistantto enzymatic degradation in a biological system and therefore theirbioavalability is improved.

The objects of the invention include remedying the deficiencies of thebackground art.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the structural similarities between a pentaerythritol (Pet)unit and a pentose and/or hexose.

FIG. 2 shows the general lipid A structures of lipopoly-saccharides fromGram-negative bacteria. Lipid A consists of a beta-(1,6)-linkedD-glucosamine disaccharide phosphorylated at 1-O— and 4′-O-positions,with numerous fatty acyl groups linked to the hydroxyl and amino groupsof the disaccharide backbone. Structure A (Imoto et al, 1985) wasisolated from E. coli and structure B (Seydel et al. 1984) was isolatedfrom Salmonella minnesota.

FIG. 3 shows a few structures derived from Pet and di-Pet units to mimicthe beta-1,6-diglucosamine disaccharide of lipid A. In structure I-IV,the glucosamine at the reducing end of lipid A disaccharide has beenreplaced by one Pet or PetNH₂ unit; in structure V-VI, the glucosamineunit at non-reducing end has been replaced by one Pet or PetNH₂ unit;and in structure VII-IX, the whole di-glucosamine disaccharide has beenreplaced by di-Pet, di-PetNH₂, or Pet-PetNH₂ unit. In structure III andIV, the non-reducing end glucosamine of lipid A disaccharide is alsoreplaced by glucose. In order to demonstrate the rationale of thedesign, a few lipid A mimics (FIGS. 4 and 5) have been prepared based onstructure I, VII and IX.

FIG. 4 shows examples of lipid structures that can be incorporated intolipid A molecules. Lipid A molecules carrying both naturally occurringlipids or un-natural lipids are well-tolerated by its binding receptor(Toll-like receptor 4) involved in immune stimulation. Lipid length isalso variable, but most preferably in the range of 12 to 16 carbons.Those fatty acyl groups shown in FIG. 4 can be attached to both hydroxyland amino groups of lipid A disaccharide backbone, while the alky groupis preferably attached to a hydroxyl group through an ether linkage.

FIG. 5 shows four lipid A mimics (1-4) prepared as examples. Both 1 and2 are designed as close structural mimics of natural lipid A (compound Aor B, FIG. 2). Compound 2 retains both phosphoryl groups of naturallipid A while compound 1 represents 1-O-de-phosphorylated analog. Inaddition, the PetNH₂ unit in 1 retains its non-chiral property while thePetNH₂ in 2 becomes chiral, which ultimately results in the formation ofa diastereomeric mixture of 2 if not separated. Structure 3 and 4 arederived from di-pentaerythritol (di-Pet) unit, carrying two phosphorylgroups but with fewer numbers of lipid chains.

FIG. 6 describes the synthesis of glycosylation donor 12 with benzylprotected phosphate group at 4-O-position. The coupling of 6 with lipidacid 7 afforded 8 in high yield. Selective opening of benzylidene ringin 8 using sodium cyanoborohydride and dry HCl (g) gave compound 9 ingood yield. Benzyl protected phosphate group was then introduced into4-O-position to form 10 in 86% yield. De-allylation followed by thereaction with trichloroacetonitrile and DBU provided the glycosylationdonor 12.

FIG. 7 describes the synthesis of glycosylation acceptor 18, a Petderivative. Benzyl substituted pentaerythritol 13 was prepared accordingto a literature procedure (Dunn et al, 1990). Dimethyl acetal formationfrom 13 gave the mono-hydroxyl compound 14, which was converted to itstosylate derivative 15. Reaction of 15 with sodium azide in the presenceof phase transfer catalyst ALIQUAT™ provided azido-substitutedintermediate 16, which was reduced to its free amine and then reactedwith trichloroethoxycarbonyl chloroformate to give 17. The removal ofthe dimethyl acetal protecting group provided the di-hydroxyl compound18 as a glycosylation acceptor for the preparation of designed lipid Amimic 1 and 2.

FIG. 8 describes thP synthesis of intermediate 20. The glycosylationreaction of 12 with excess 18 (4.0 eq.) gave the desiredmono-glycosylated product 19 in the presence of TMSOTf as catalyst in81% yield. Treatment of 19 with zinc powder in acetic acid resulted inthe removal of both Troc-group to give di-amine intermediate, which wasthen coupled with lipid acid 7 to provide intermediate 20. ¹H NMRspectrum data of 20 showed two sets of doublet at d 4.35 (J=8.0 Hz) andd 4.65 (J=8.0 Hz), which confirmed the presence of two diastero-isomersin about 1:1 ratio, with both having b-linkage.

FIG. 9 shows the final preparation of the designed compound 1 and 2.Hydrogenolytic debenzylation of 20 in the presence of palladium oncharcoal gave 1 in quantitative yield. On the other hand, theintroduction of another benzyl-protected phosphate group into the freehydroxyl group of 20 provided 21, which was de-protected to afford thefinal product 2. The structure of both 1 and 2 were confirmed by ¹H NMRand ESIMS spectra data.

FIG. 10 describes the synthesis of lipid A mimic 3. Di-pentaerythritolwas first protected as di-benzylidene acetal 22 which was reacted withtetradecyl bromide in the presence of sodium hydride to givedi-lipidated compound 23. Reductive ring opening of benzylidene acetalsby treating 23 with sodium cyanoboronhydride and trifluoroacetic acidafforded 24 in moderate yield. Introduction of two benzyl-protectedphosphate groups into 24 gave the precursor 25 which upon the treatmentwith palladium on charcoal under hydrogen atmosphere resulted in thedesigned product 3.

FIG. 11 describes the synthesis of the intermediate 29. Compound 22 wastreated with tosyl chloride and pyridine to give the di-tosylate 26,which was converted to di-azide 27 by reacting with sodium azide in thepresence of the phase catalyst Aliquat™ 336. Azide reduction withdithiopropane afforded the di-amine compound 28, which upon the couplingwith di-lipo acid 7 provided the intermediate 29 in 66% yield.

FIG. 12 describes the synthesis of di-Pet derived lipid A mimic 4.Through the same reaction steps as described for the preparation ofcompound 3 (FIG. 10), compound 29 was converted to the target molecule 4in overall good yield.

FIG. 13 shows some more structures designed as lipid A mimics (X-XII)containing PetNH₂ and di-PetNH₂. Structure X and XI are based on di-Petskeleton with unsymmetrical lipid distribution. In structure XII, thePetNH₂ unit has replaced the non-reducing-end glucosamine of the lipid Adisaccharide.

FIG. 14 exhibits the potency of lipid A mimic 1 and 2 in inducing the invitro secretion of cytokines by adherent cells isolated from humanperipheral blood. R595 lipid A, a natural lipid A product isolated fromSalmonella minnesota, R595. (Avanti Polar Lipids, Inc.), was also testedalong for comparison. The secretion pattern was determined for (a)secretion of tumor-necrosis factor-alpha (TNF-alpha, pg/mL); (b)secretion of IL-6 (pg/mL); (c) secretion of IL-8 (pg/mL). The data showsthat lipid A mimic 1 and 2 are comparable to R595 lipid A in activationof secretion of all three cytokines, TNF-alpha, IL-6 and IL-8. It isquite reasonable to believe that mimic 1 and 2 activate these humanmonocytes by similar mechanism as their natural counterparts.

FIG. 15 shows the induction of antigen specific T cell proliferationresponse by synthetic liposomal vaccine BLP25 containing lipid A mimic 1or 2 as an adjuvant. A MUC1 derived 25-mer lipopeptide,H₂N-STAPPAHGVTSAPDTRPAPGSTAPPK (palmitoyl) G-OH, was used as theantigen. T cell proliferation data presented in FIG. 15 clearlydemonstrates that C57BL/6 mice immunized with one dose of BLP25liposomal vaccine produces a potent T cell response specific to MUC1antigen. The response in the mice immunized with liposomal formulationcontaining synthetic lipid A mimic 1 or 2 is comparable to that in thegroup of mice immunized with formulation containing R595 lipid A. Whenthe liposomal formulation contains no lipid A analog as an adjuvant, theantigen specific T cell proliferation response is very low (data notshown).

FIG. 16 shows the inhibitory effect on tumor growth of a liposomalvaccine containing a lipid A analog as an adjuvant. The liposomalvaccine BLP25 contains a MUC1 derived 25-mer lipopeptide and lipid Amimic 1 or 2, or R595 lipid A. Active specific immunotherapy of MC-38MUC1 tumor bearing mice was performed by immunizing intradermally withBLP25 liposomal formulation. Mice were challenged with tumor on day 0and immunized on day 7, 14 and 21. On day 34, tumor diameters (length &width) were taken and tumor size was expressed as mm² (length width). Aspresented in FIG. 16, BLP25 liposomal vaccine adjuvanted with syntheticlipid A mimic 1 or 2 produces tumor inhibition effect comparable to thatproduced by BLP25 formulation adjuvanted with R595 lipid A. In thecontrol group of mice immunized with saline alone, tumor size is aboutthe double of those immunized with BLP25 vaccine adjuvanted with lipid Amimic 1 or 2.

FIG. 17 shows a synthetic strategy for preparing one PetNH₂-containingcarbohydrate mimic, TM. TM is based on the terminal tetrasaccharide ofthe tumor-associated Globo-H antigen in which the N-acetyl-galactosamineis replaced by PetNHAc. One arm of the PetNH₂-core is linked to thereducing-end galactose through an ether linkage while another arm islinked to the disaccharide through a glycosidic bond. Differentmethodologies are employed to construct this two different types ofbonds. The ether bond may be constructed by classical S_(N)1/S_(N)2substitution reaction while the glycosidic bond can be constructedthrough glycosylation reactions by using various kinds of glycosylationdonor (e.g. trichloroacetimidate method as shown in FIG. 17). Standardprotecting group manipulation, step-wide coupling, and finaldeprotection would result in the fully deprotected product TM.

FIG. 18 shows the structure of BLP25 lipopeptide (SEQ ID NO:1) derivedfrom MUC1 mucin. The lipopeptide is a synthetic tumor-associated antigenused for the biological evaluation of lipid A mimic 1 and 2. A liposomalformulation containing BLP25 lipopeptide and either lipid A mimic 1 or 2shows therapeutic effect in inhibiting tumor growth in mice.

FIG. 19 shows some examples of PetNH₂-containing new structures derivedfrom tumor-associated carbohydrate antigens. These carbohydrates areassociated with cancer progression and are expressed at higher level oncancer cells than on normal cells. Great efforts have been made todevelop potential therapeutic agents for cancer treatment from thesecarbohydrates. (S. J. Danishefsky & J. R. Allen, Angew. Chem. Int. Ed.2000, 39, 836-863). Structural mimetics are expected to exert similarimmunological significance. Immune responses directed toward thesemimetic structures are deemed to recognize their natural counterparts.For example, antibodies raised against the mimetic structure (TM, FIG.17) of the Globo-H terminal tetrasaccharide is expected to cross-reactwith the cancer cells expressing Globo-H antigen.

FIG. 20 shows some new structures derived from those carbohydratesinvolved in the event of bacterial adhesion onto host cells.Glycosphingolipids (e.g., GM1, GM2, and GM3) and Lewis seriescarbohydrates (e.g., Le^(a), Le^(b), Le^(x), Le^(y), sialyl Le^(x),etc.) are well known to play important roles in bacterial colonizationonto host cells. It is general believed that molecules that inhibit thiscolonization process can be effective anti-bacterial agents in that theyprevent the entry of bacteria to the host. Carbohydrate ligands in itsnatural form are poor inhibitors due to their low binding constants andtheir instability toward enzymatic degradation. Thus, synthetic mimeticsof these natural carbohydrate ligands offer an opportunity to improvetheir low binding constant and low bioavailability in the biologicalsystem. For exmaple, H type I blood determinant trisaccharide (FIG. 20)is implicated in adhesion involving the pathogenic bacteria Helicobacterpylori. Its structural mimetic provides an alternative skeleton wherefurther chemical modifications can be maneuvered in order to find newmolecules with higher inhibition efficiency.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION

For the sake of clarity, it should be noted that when the abbreviation“Pet” is used in the context of a structural component of the lipid Aanalogs of the present invention, what is intended is the “residue” ofpentaerythritol, i.e., pentaerythritol less one or more of itshydrogens, so that it can be incorporated into a larger chemical entity.Moreover, the term “Pet”, when used in this context, includes thedisclosed modified moieties which retain the Pet five carbon core(2,2-dimethylpropane), but in which one or more of the hydroxyloxygensis replaced with a spacer moiety Yl-Y4 as defined below.

Likewise, when the abbreviation “Pet-NH2” is used in the context of astructural component of the carbohydrate ligand analogs (including lipidA analogs) of the present invention, what is intended is the “residue”of pentaerythritamine, i.e., the latter less one of the amino hydrogens,and optionally less one or more of the hydroxyl hydrogens. Moreover, theterm “Pet-NH2”, when used in this context, includes the disclosedmodified moieties in which one or more of the hydroxyloxygens arereplaced with a spacer moiety Y1-Y4 as defined below. The symbol

Pet-NH— is sometimes used to indicate that the amino function must bepresent, but that the other functions are subject to modification.

It should further be evident that the term “Pet” includes “Pet-NH2” as aspecial case, i.e., one in which one hydroxyl oxygen is replaced bynitrogen. If it is necessary to refer specifically to the situation inwhich none of the Pet carbons is aminated, one may use “Pet-OH” or“Pet-chal”, the “chal” denoting chalcogen.

Lipid A Analogs

Bacterial Lipid-A compositions are widely used as adjuvants to enhancethe immune responses to various antigens used in vaccine formulations.

The present invention relates to novel synthetic structural analogs ofbacterial Lipid-A, especially E. coli lipid A, and methods of synthesisof such analogs. These lipid A analogs may be agonists or antagonists ofbacterial lipid A. Agonists are likely to have a higher degree ofstructural similarity to lipid A than are antagonists.

Synthetic Lipid-A analogs have several advantages over naturally derivedadjuvant preparations. A synthetic compound is chemically defined withsingle structure and thus facilitates its tracking and control frommanufacturing to final formulation. Synthetic product is cost effectiveand is easily adaptable for commercial scale-up while maintaining theconsistency in both quality and performance.

An invariant structural feature of the natural E. coli Lipid-A moleculeis its β-(1,6)-linked D-glucosamine disaccharide backbone. However, ithas been shown that monosacharide analogs can express endotoxicactivities. See, e.g., Matsuura, et al., Infect. & Immun. 63: 1446-51(1995); Funatogawa, et al., Infect. & Immun., 66: 5792-98 (1998).Moreover, lipid A agonists are known in which the entire disaccharideunit has been replaced with an acyclic backbone, see Hawkins, J.Pharmacol. Exp. Therap. 300: 655-61 (2002).

The Lipid A analogs of the present invention replace at least one of thesugar units of natural Lipid A with the five carbon backbone (core) ofpentaerythritol (Pet). This features a central carbon, singly bonded tofour peripheral carbons:

These carbons are, in turn, be joined to other moieties.

The remaining sugar unit may be retained (possibly in a modified form),likewise replaced with Pet, or omitted altogether.

Thus, the lipid A analogs of the present invention have the structure

where Al-A4 are hereafter defined. Each of Al-A4 may be considered a“primary branch” of the analog. Note that none of Al-A4 are merelyhydrogen.

Since conservation of the sugar units is not considered important, oneor both sugar units of lipid A may be replaced in the analog by a Petunit, or one may be so replaced and the other omitted withoutreplacement.

In general, to preserve structural similarity to lipid A, the followingfurther limitations apply to Al-A4:

(1) at least one of Al-A4 comprises at least one phosphate equivalent (aphosphate group, or an analog thereof as described below), and(2) at least one of Al-A4 comprises at least one strongly lipophilicgroup as defined below.

With regard to limitation (1), natural lipid A is diphosphorylated, butit is known that the monophosphorylated analog is active, and applicantsbelieve that certain phosphate analogs will also be efficacious.

With regard to limitation (2), natural lipid A is, plainly, lipidated,and if delipidated loses its immunostimulatory activity.

In a preferred embodiment, A₁ is Y₁R₁, A₂ is Y₂R₂, A₃ is Y₃R₃ and A₄, isY₄R₄, where Y₁-Y₄ are spacers as hereafter defined. Preferably, each ofR₁-R₄ is, independently, selected from the group consisting of hydrogen,an organic group, or a group which in conjunction with the adjacent Ygroup forms a phosphate, sulfate or borate. To put it another way,preferably each of R1-R4 is independently selected from the groupconsisting of hydrogen, —P(═O) (OH)OH, —C(═O)OH, —S(═O) (═O)OH,—B(OH)OH, or

an organic group. Preferably, each of these organic groups has not morethan 200 atoms other than hydrogen, more preferably, not more than 150,still more preferably, not more than 100.

The Pet unit may be considered to be the Pet backbone (core) as definedabove, together with the Y₁-Y₄ groups which correspond to or replace thehydroxyloxygens of unmodified Pet:

It is further noted that there may be more Pet units in the analog thanthose used to replace one or both sugar units of lipid A. Such “extra”Pet units may be useful as scaffolds for the attachment ofphosphate-equivalents, strongly lipophilic groups, and other usefulchemical moieties. Preferably, there are not more than two “extra” Petunits (i.e., a total of three Pet units if the analog includes a sugarunit, or a total of four Pet units if it doesn't). More preferably,there is just one, and most preferably, there is no “extra” Pet unit.

When there are two or more Pet units in an, analog, they may beadjacent, or separated by another moiety. If they are adjacent, then oneof the spacers Yl-Y4 of one Pet unit serves also as one of the spacersYl-Y4 of the adjacent Pet unit, as seen, for example, in FIG. 3,compounds VII-IX, and FIG. 5, compounds 3 and 4.

Alternatively, there may be another chemical moiety connecting thespacer of one Pet unit and the spacer of the other Pet unit. This moietymay, but need not, comprise a sugar unit, a strongly lipophilic group,and/or a phosphate equivalent.

When there are more than two Pet units in an analog, they may beconnected linearly (Pet1 . . . Pet2 . . . Pet3), cyclically (Pet1 . . .Pet2 . . . Pet3 . . . Pet1), or in a branched form (Pet1 . . . Pet2( . .. Pet3) . . . Pet4), or in some combination thereof. Note that in theabove, “ . . . ” denotes a connection that may be adjacent or throughsome other chemical moiety.

In a preferred embodiment, if the lipid A analog comprises a sugar unit,the lipid analog is one such that if prepared as a thin multilayer filmas described by Seydel et al., Eur. J. Biochem. 267: 3032-39 (2000), thetilt angle of the sugar backbone of the analog relative to the“membrane” surface, determined as taught by Seydel, is at least 35°,more preferably over 50°, if an agonist is sought, and the tilt angle isless than 25° if an antagonist is desired. It must be emphasized thatthis is merely a preferred embodiment and it is not necessary that thetilt angle be determined, or, if determined, that it be in the rangessuggested above.

Primary Arms

The numbering of the primary arms Al-A4 and their components Yl-Y4 andR1-R4 is completely arbitrary.

One approach to classifying the analogs is one the basis of whether theyprovide one sugar unit, a second Pet core, or neither.

Another approach to classification is on the basis of the number of theR groups R1-R4 which are H.

In a first class of analogs, R1—R3 are H, and R4 comprises the stronglylipophilic group(s), the phosphate equivalent, and, optionally, thesugar unit or second Pet core. In this class, it is preferable thateither all of Al-A3 be —OH, or that two be —OH and the third —NH₂. InR4, the component proximal to the Pet core may be the stronglylipophilic group, the phosphate equivalent, or, if present, the sugarunit or second Pet core. Preferably, R4 includes a sugar or second Petcore, and more preferably this is the component proximal to the firstPet core, and the phosphate equivalent and at least one stronglylipophilic group are connected to it.

In a second class of analogs, just two of the R1-R4 are H (andpreferably the corresponding Y groups are —O—), and therefore thestrongly lipophilic group, the phosphate equivalent, and optionally, thesugar unit or second Pet core, are distributed among the remaining twoarms. Thus, in compound 1, one arm consists of an NH linked stronglylipophilic group, and a second consists of an O-linked phosphated andlipidated sugar unit.

In a third class of analogs, just one of the R1-R4 is H (and preferablythe corresponding Y group is —O—), and the strongly lipophilic group(s),the phosphate equivalent(s), and, optionally, the sugar unit or secondPet core, are distributed among the remaining three arms.

Thus, in compound 2, one arm is phosphate (note that one of thephosphate oxygens does double duty as the Y group), a second arm is anNH-linked strongly lipophilic group, and the final arm is an O-linkedsugar which is both lipidated (through —NH—) and phosphated. Compound 4is similar, except that it is a second Pet core, rather than a sugarunit, which is lipidated and phosphated. Compound 3 differs from 4 inthat the lipid is O-rather than NH-linked to the Pet cores.

In a fourth class of analogs, none of R1-R4 is H. Since at most one armcan comprise a sugar unit or a second Pet core, this implies that theother three arms comprise phosphate equivalents and/or stronglylipophilic groups. And that in turn implies that there must be at leasttwo phosphate equivalents or at least two strongly lipophilic groups.

A, third approach to classification is on the basis of the number andlocation of the phosphate equivalents. The classes are then (1) onephosphate equivalent, (2) two phosphate equivalents, but on the samearm, (3) two phosphate equivalents, on different arms, or (3) more thantwo phosphate equivalents. We may further subdivide them on the basis ofwhether phosphate equivalent is connected to a sugar unit or not.

A fourth approach to classification is on the basis of the number andlocation of the strongly lipophilic groups. For example, in compounds 1and 2 there are three strongly lipophilic groups, and in compounds 3 and4 there are two. Also, they may be connected to the Pet core (through aY spacer), to a sugar unit, or to a phosphate equivalent.

Connection of Major Elements

In this specification, four major elements of the lipid A analog aredefined: a Pet unit, a sugar unit, a strongly lipophilic group, and aphosphate equivalent. When it is said that two major elements areconnected, it means without any other major element intervening. Theremay be some other chemical moiety, such as the disclosed linkers andspacers, in-between them.

Thus, when it is said that a strongly lipophilic group is connected to aphosphate equivalent, it means, without any intervening sugar unit orPet unit. Likewise, when it is said that a strongly lipophilic group isconnected to a sugar unit, it means, without any intervening Pet unit orphosphate equivalent. Conversely, when it is said that a stronglylipophilic group is connected to a Pet unit, it means, without anyintervening sugar unit or phosphate equivalent. Analogous examples canbe given for the other possible two-way connections of four kinds ofmajor elements.

The specification may also identify two elements as being linked by athird element, in which case each of the former elements are connectedto the latter.

Spacers (Y1-Y4)

Pentaerythritol can be considered to be the compound of general formulaI in which Al-A4 are all —OH. Equivalently, it is the compound of thatformula in which Yl-Y4 are all —O— and R1-R4 are all —H.

While pentaerythritol per se is not one of the lipid A analogs of thepresent invention, the latter does contemplate the incorporation ofspacers Yl-Y4 which are —O— or analogs thereof.

In a preferred embodiment, each of spacers Y1-Y4 is independentlyselected from the group consisting of —(CH₂)_(n)O—. —(CH₂)_(n)S—, and—(CH₂)_(n)NH—, where n is, independently, 0 to 4. More preferably, eachof these spacers is —O—, —S— or —NH— (i.e., n is 0). Even morepreferably, each of these spacers is —O— or —NH—. Most preferably,either (a) all of these spacers are —O—, or (b) one spacer is —NH— andthe other spacers are —O—.

Phosphate Equivalents in Lipid A Analogs

Natural Lipid A features two phosphate groups, each attached to a sugarunit (Lipid A being a disaccharide). However, it has been shown thatmonophosphoryl lipid A (MPLA) has adjuvanting activity and is less toxicthan the natural diphosphorylated molecule. Also, we believe that one ormore of the phosphate group(s) of lipid A and MPLA can be replaced bycertain related chemical moieties.

Hence, in the lipid A analogs of the present invention, at least one ofAl, A2, A3 and A4 comprises at least one —O—P(═O)(OH)—O—, —C(═O)0H,—O—S(═O)₂—O—, or —O-B (OH)—O— moiety, these being listed in order frommost to least preferred. A phosphate analog is here defined as such amoiety, other than phosphate itself. A phosphate equivalent is heredefined to include both phosphate and the phosphate analogs.

Preferably, if the lipid A analog lacks any sugar unit, at least onephosphate equivalent comprises a —O—P(═O)(OH)—O—, —O—S(═O)₂—O—, or—O—B(OH)—O— moiety.

The three aforementioned structures can be used to link the Pet core toa chemical moiety comprising at least one strongly lipophilic group,and/or a sugar equivalent selected from the group consisting of a sugarunit and a second Pet core. In such instance, one of the —O—'s of thephosphate equivalent is deemed the spacer Yl-Y4 referred to elsewhere.

Alternatively, the phosphate equivalent can be essentially a terminalmoiety (the —C(═O)OH always is). Thus, in some preferred embodiments, atleast one phosphate equivalent is of the form —OB(OH)OR, —OP(═O) (OH)ORor —OS(═OH) (OH)OR, where R is hydrogen, or a substituted orunsubstituted alkyl group of 1-4 carbons. If R is hydrogen, then threeof these moieties reduce to inorganic moieties: borate, phosphate andsulfate. If R is a substituted group, then the substitutions arepreferably —OH or —NH2. An R group of particular interest is CH₂CH₂NH₂.Another structure of interest is —OP(═O)(OH)—O—P(═O)(—OH)—O—R, disclosedby Ulmer.

In other preferred embodiments, at least one phosphate equivalent is ofthe form —R′—C(O)OH, where R′ is a substituted or unsubstituted alkylgroup of 1-4 carbons. More preferably, R′ is —CH₂—.

The lipid A analogs of the present invention preferably have one or twophosphate equivalents, and if they have more than one, they may be thesame or different. Thus, they could have one phosphate and one phosphateanalog. If there is more than one, the phosphate equivalents may beincorporated into the same or, more preferably, different primarybranches of the analog.

In some preferred embodiments, at least one of Al-A4 will be thephosphate equivalent. In that case, one of the oxygens of the phosphateequivalents also serves as the Yl-Y4 spacer for that arm.

In other preferred embodiments, the phosphate equivalent will be asubstituent of a larger moiety which connects to the aforementionedspacer. In an especially preferred sub-embodiment, this larger moiety isthe aforementioned sugar or sugar analog, as, in natural lipid A, thephosphate group is attached to a sugar unit. In E. coli lipid A,phosphate groups are attached to the C-1 and C-4′ carbons of the coredisaccharide.

In still other preferred embodiments, at least one phosphate equivalentis incorporated into at least one of the aforementioned lipophilicgroups.

Optional Sugar Unit of Lipid A Analogs

It should be noted that natural lipid A is a disaccharide, and therequired Pet unit of the analog replaces one of the two sugar units ofthat disaccharide. Hence, the lipid A analog will have either one or nosugar units. If it has no sugar units, it is because the second sugarunit of natural lipid A was replaced by a Pet core, or was omittedaltogether.

If the analog includes a sugar, it need not be the same sugar as innative lipid A, i.e., a glucosamine. However, it is preferable that itbe a hexose and/or a cyclic sugar (especially a pyranose), and morepreferable that it be a glucose or glucose derivative, and still morepreferable that it be a glucosamine.

If the analog comprises only one Pet unit, then preferably the phosphateequivalent is not —COOH, and preferably the analog does not comprise anynucleobase.

Lipid Complement of Lipid A Analogs

Lipid diversity contributes to by far the most significant variationsamong natural Lipid-A structures. While they are all linked throughester and amide bonds to the hydroxy and amino groups of the sugarrespectively, variations include the number of lipids attached, thelength of each lipid chain and the functional groups contained withinthe lipid chains. It is believed that these variations contribute tovarious biological functions of the entire Lipid-A molecule and moreimportantly to its adjuvant properties.

In some preferred embodiments, the lipid A analog comprises at least onestrongly lipophilic group which is identical to a lipid chain occurringin a natural Lipid A structure. In a sub-embodiment, all of the stronglylipophilic groups of the lipid A analog are groups which occur innatural Lipid A structures, but it is not required that they all occurin the same natural lipid A molecule, or even in the contingent oflipids found in the natural lipid A molecules of the same bacterium.However, these further restrictions may be considered furthersub-embodiments.

A major advantage provided by the synthesis of a Lipid-A analog is thata molecule may be designed to achieve effectiveness as an adjuvant,safety and stability by modifying lipid chains and their linkages.

Hence, in other preferred embodiments, the lipid A analog comprises atleast one strongly lipophilic group which is not found in any naturalLipid A structure. The difference may be, but is not limited to, adifference in the length of the chain, the degree of branching of thechain, the presence or location of unsaturated linkages, or the presenceor location of —COO— (ester), —O— (ether) or —NH— (amino) linkages.

Chemically speaking, ester linkages are labile as they are vulnerable tohydrolysis under physiological conditions. Gradual loss of lipid chainsmay slowly reduce the activity of the adjuvant under long storage of thevaccines thus diminishing their shelf life. Introduction of unnaturalbut stable ether linkages in place of esters may therefore beadvantageous.

In the major form of natural E. coli lipid A, the discaccharide backboneis composed of two glucosamines, which we will call sugar II (it has aphosphate on the 4′ carbon) and sugar I (it has a phosphate on the 1carbon). The lipid component takes the form of six carbon chains, linkedto the 2′ and 3′ carbons of sugar II and the 2 and 3 carbons of sugar I.

A branched lipid, is O-linked to the 3′-carbon. A similar branched lipidis N-linked to the 2′ carbon. In both branched lipids, the primary chain(the one linked to the sugar ring carbon) is an acyl chain. A secondaryacyl chain is O-linked to the C-3 carbon of the primary acyl chain (thecarbonyl carbon being C-1). Thus, a total of four carbon (acyl) chainsare linked directly or indirectly to sugar II.

Additionally, an unbranched but hydroxylated acyl chain is O-linked tothe 3 carbon of the sugar ring and another such acyl chain is N-linkedto the 2 carbon of the sugar ring. Thus, a total of two carbon (acyl)chains are linked to sugar I.

Since there are four acyl chains on one sugar, and two on the other,purified E. coli lipid A (Alexander, 2002, FIG. 2A; Se3ydel, 2000, FIG.1A, “hexaacyl lipid A”) is said to have an asymmetric hexaacyl lipidcomplement, and, more specifically, a 4/2 distribution. (All referencesto “lipid A” are, unless qualified, to this purified E. coli lipid A asdescribed above.)

The lipid complement of the present Lipid A analogs consists essentiallyof one or more strongly lipophilic groups as defined in a later section.Each strongly lipophilic group preferably provides one or more majorcarbon chains as hereafter defined. Collectively, the lipid complementof the present lipid A analogs preferably provides one, two, three,four, five, six, seven, eight or more major carbon chains, with three tosix being most preferred. Preferably, each strongly lipophilic groupprovides one, two or three major carbon chains. Preferably, these majorcarbon chains are each 10-20, more preferably 12-16 carbons.

In E. coli lipid A, the lipid groups provide 82 carbon atoms, and in S.minnesota lipid A, 98 carbons (7 acyl chains), while in R. capsulatuslipid A, which is an endotoxin antagonist, they provide 60 carbon atoms.There are monosaccharide analog lipid A agonists whose lipid groupsprovide 42 carbon atoms.

Hence, preferably, the major carbon chains of the strongly lipophilicgroups collectively provide at least 20, at least 30, at least 40, atleast 50, at least 60, at least 70, or at least 80 carbon atoms.Desirably, they provide not more than 120, not more than 110, not morethan 100, not more than 90, not more than 80, not more than 70 or notmore than 60.

Preferably the sum of the predicted log Ps (see below) for the stronglylipophilic groups is at least 10, at least 15, at least 20, at least 25,at least 30, at least 40, or at least 50. Preferably, it is not morethan 60, not more than 40, not more than 40 or not more than 30.

Each strongly lipophilic group is preferably connected to the remainderof the analog by a proximal linker selected from the group consisting of—O—, —S—, and —NH—.

It may be so connected to the carbon of a sugar or Pet core, or to thesulfur, phosphorus or boron atom of a divalent phosphate equivalent. Inthe case of connection to a sugar, the proximal linker is the oxygen ofa sugar hydroxyl, the sulfur of a thio sugar, or the nitrogen of anamino sugar. In the case of connection to the Pet core, the proximallinker is a portion of the spacer Yl-Y4. In the case of connection tothe aforementioned atom of a phosphate equivalent, the proximal linkeris an —O— of said phosphate equivalent.

This proximal linker may be bonded directly to a major carbon chain asdefined below, or to a distal linker. The distal linker may be divalent,trivalent, tetravalent, etc. Usually it will be at least trivalent, thusserving to connect the remainder of the analog to at least two differentmajor carbon chains of the lipophilic group. The distal linker consistsof two or more elements independently selected from the group consistingof alkyl of 1-5 carbon atoms, —O—, —S—, —C(═O)—, —C(═S)—, —NH—, and —N<,with the caveat that the atoms of the distal linker connected directlyto the major carbon chains of the lipophilic group are not carbon atoms(if they were, then those atoms would be part of the carbon chain, notpart of the distal linker).

In FIG. 4, the seventh and eighth structures feature distal linkers. Inthe seventh structure, it is the trivalent —C(═O)—CH(—CH₂—O—)—CH₂—O—. Inthe eighth structure, it is the trivalent —C(═O)—CH2—CH(—NH—)—C(═O)O—.

For the purpose of determining whether a group attached to a sugar is astrongly lipophilic group, the proximal linker is disregarded, but thedistal linker is considered part of the group. Likewise, for a groupattached to the Pet core, the intervening spacer is disregarded.

If the lipid A analog provides a sugar, at least one of the followingsites on the sugar carbon skeleton may be linked to a stronglylipophilic group:

(A) the anomeric ring carbon(B) the other ring carbon immediately adjacent to the ring heteroatom(usually oxygen)(C) a ring carbon other than those of (A) or (B) above(D) a sugar carbon other than a ring carbon.It will be understood that such linkage will usually be through a linkersuch as the “proximal linker” defined herein, but a connection without alinker (i.e., a C-substituted amino acid) is not absolutely excluded.

If the sugar is a hexose and a pyranose, like glucose, at least one ofthe following sites may be linked to a strongly lipophilic group:

(1) the C-2 or C-2′ carbon of the sugar rings (i.e., one of the sites atwhich natural lipid A is N-lipidated);(2) the C-3 or C-3′ carbon of the sugar rings (i.e., one of the sites atwhich natural lipid A is O-lipidated);(3) the C-1′ (anomeric) carbon of the sugar II ring (in natural lipid A,this carbon is linked to the C-6 of the sugar I, but if the sugar I isomitted, then this carbon is free);(4) the C-1 (anomeric carbon) of the sugar I ring (in natural lipid A,this carbon is phosphorylated);(5) the C-6 non-ring carbon of the sugar I (in natural lipid A, thiscarbon is linked to the C-1 of the sugar II, but if the sugar II isomitted, then this carbon is free);(6) the C-6′ non-ring carbon of the sugar II (in the lipid Adisaccharide based on natural lipid A, this bears just —OH, but this isnormally the site of attachment of the lipid A disaccharide to theremainder of the LPS molecule);(7) the C-4′ carbon of the sugar II ring (in natural lipid A, this isphosphated);(8) the C-4 carbon of the sugar I ring (in natural lipid A, this bears afree hydroxyl).

Preferably, the strongly lipophilic groups are attached to the C-2 andC-3 of sugar I and the C-2′ and C-3′ of sugar II.

The use of a phosphate linker satisfies the requirement for a phosphateequivalent, but other phosphate equivalents may be provided, if desired.The use of a phosphate linker is preferred in the case of substitutionsat the 4′ ring carbon.

The —O— linker is preferred at the 4, 3 and 3′ carbons, and the —NH—linker at the 2 and 2′ carbons. It should be appreciated that if the NH2group on these carbons is lipidated, the NH2 becomes an NH linker.Likewise, if the 4-OH is lipidated, the —OH becomes an —O— linker.

There is no particular preference with regard to the linker at theanomeric carbon or at the non-ring carbons of the sugar.

Alternatively or additionally, at least one lipophilic group may beincorporated into one or more of the primary arms of the Pet unit,without becoming a substituent of the sugar unit, if any. The primaryarm in question may consist essentially of the strongly lipophilicgroup.

The strongly lipophilic group will in general comprise one or morecarbon chains. Each carbon chain will be composed of carbon atoms linkedsequentially by single, double or triple bonds.

Carbon chains which are at least six carbons in length are considered“major” carbon chains. Other carbon chain are considered “minor” carbonchains. The strongly lipophilic group preferably comprises at least onemajor carbon chain. There is no preference one way or another as to thepresence of minor carbon chains.

Minor carbon chains can be considered a species of linker. In theseventh and eighth structures in FIG. 4, there are minor chains.

Preferably, no more than one bond of a particular carbon chain is adouble or triple bond, and more preferably, the carbon chain is fullysaturated. Double bonds are preferred over triple bonds.

The carbon atoms of a carbon chain may be bonded to 3, 2, 1 or 0hydrogens. In a major carbon chain, the —CH< and >C< carbons are usuallybranching points for the attachment (with or without a linker) ofanother carbon chain. They may also be substituted with a side group,such as amino or hydroxyl.

Purely as a matter of definition, the strongly lipophilic group cannotcomprise a Pet unit (it may comprise a Pet core if it lacks one or moreof the required spacers Y1-Y4). However, what might otherwise have beeninterpreted as one large strongly lipophilic group comprising a Pet unitmay be reinterpreted as a Pet unit with one or more smaller stronglylipophilic groups attached to it.

The carbon atoms of any major carbon chain may include one or morecarbonyl or thiocarbonyl carbons, i.e., —C(═O)— or —C(═S)—. Carbonyl ispreferred. If there is only one carbonyl or thiocarbonyl carbon, it ispreferably at the beginning of the chain, so the chain is an acyl chain(saturated or unsaturated). Thus, if the linker is —O—, the attachmentto carbonyl forms an ester (—O—(C═O)—), and if it is —NH—, theattachment forms an amide (—NH—(C═O)—.

A particular lipophilic group may be a simple (unbranched, acyclic)lipid, or a complex (branched and/or cyclic, including partiallyaromatic) lipid.

If the lipophilic group comprises more than one major carbon chain, themajor chain beginning closest to the sugar or pet core is considered theprimary major chain of the group. Any chains attached to the primarymajor chain are considered secondary major chains. Any major chainsattached to the secondary major chains are considered tertiary majorchains, etc. (Reference to primary, secondary, etc. chains hereafter isto major chains unless otherwise indicated.)

It is possible that several major chains will be equally close to thesugar or Pet core, in which case they will each be primary chains.

A secondary chain may be attached to the distal end (relative to thesugar or Pet core) of the primary chain, in which case the lipophilicgroup remains linear (absent other moieties). Or it may be attached toan interior carbon of the primary chain, in which case the lipophilicgroup is a branched lipid.

A secondary chain may be attached to a primary chain by a simple —O—,—S— or —NH— linker, or it may be attached directly without a linker(i.e., C—C). It also may be attached by a complex linker, i.e., acombination of a simple linker and the distal linker previously defined.A tertiary chain may be attached to a secondary chain in the samemanner, and so on. A preferred point of attachment of a higher orderchain to a lower order chain (e.g. secondary to primary) is at the C-3carbon of the lower order (e.g., primary) chain.

Like a primary chain, a secondary or higher order chain may comprisedoubly or triply bonded carbon atoms, and/or carbonyl or thiocarbonylcarbons.

The various carbon chains referred to above may be substituted withhydroxyl or amino groups, with hydroxyl being preferred. Preferredpositions for the hydroxyl group would be as substituents on the C-2 orC-3 carbon of the chain.

The strongly lipophilic group may be entirely aliphatic or it may bepartially aromatic in character. If it includes an aromatic structure,that structure is deemed a separate major carbon chain even if directlyattached to an aliphatic chain. An entirely aliphatic group ispreferred.

Fatty acid groups of the form —O—CO-Q, where Q is primarily alkyl butmay include alkenyl, alkynyl, or ether linkages, are of particularinterest. The fatty acids are carboxylic acids, often derived from orcontained in an animal or vegetable fat or oil. All fatty acids arecomposed of a chain of hydrocarbon groups containing from 4 to 22 carbonatoms and characterized by a terminal carboxyl radical. They may bedesignated by “the number of carbon atoms: number of double bonds”, andoptionally the locations of cis/trans isomerism. Thus, suitable fattyacids include those with designations 4:0, 6:0, 8:0, 10:0, 12:0, 14:0,16:0, 16:1 (9c), 18:0, 18:1 (9c), 18:2 (9c, 12c), 18:3 (9c, 12c, 15c),18:4 (6c, 9c, 12c, 15c), 18:3 (9c, llt, 13t), 18:1 (9c) 12-OH, 20:1(9c), 20:1 (11c), 20:4 (8c, llc, 14c, 17c), 20:5 (5c, 8c, llc, 14c,17c), 22:0, 22:1 (11c), 22:1 (13c), 22:5 (7c, 10c, 13c, 16c, 19c) and22:6 (4c, 7c, 10c, 13c, 16c, 19c), all of which are found in naturallyoccurring glycosides.

The lipid structures which occur in natural lipid A from various speciesinclude 10:0, 12:0, 14:0, 16:0, 18:0, 20:0 fatty acids. Secondary acylgroups are usually 3-O-attached. Hydroxylation is usually 3-OH or 2-OH.A number of lipid As (e.g., Rhodobacter capsulatus and Rhodobactersphaeroides) include 12:1 of 14:1 secondary acyl groups. See Alexander,et al., Trends in Glycoscience and Glycotechnology, 14: 69-86 (March2002).

In a preferred embodiment, at least one strongly lipophilic group of thelipid A analog is a strongly lipophilic group not used as a protectinggroup in carbohydrate synthesis. Protecting groups used in carbohydratesynthesis include methyl, benzyl, allyl, trityl (triphenylmethyl),various acetates, benzoate, etc. Benzylidene and isopropylideneprotecting groups may simultaneously protect two adjacenthydroxyloxygens. See generally Harwood, Modern Methods in CarbohydrateSynthesis (1996); Dekker, Preparative Carbohydrate Chemistry (1997);Blackie, Carbohydrate Chemistry (1998).

The following generic structures are of interest:

where X is —CO— or —CH₂₋, k is an integer 4-30;

where n is an integer 0-6, k is an integer 0-30 and 2k+3n is an integer4-30;

where m and n are integers (0-6 for n and 0-30 for m), and m+n+1 is4-30;

where m+n+1 is 4-30;

where X₁ and X₂ are independently —CO— or —CH_(2—), and m+n+k+1 is 4-30;

Where Z is —NH— or —O—, and k+m+2 is 4-30.

where q is an integer 0-6, and k+q+m+n is 4-30.

where X₁, X₂, and X₃ are independently —CO— or —CH₂—, r is an integer0-6, and r+k+q+m+n is 5-30.

In each of cases (i)-(viii), previously defined parameters retain theirmeaning.

See also the lipid A analog substituents suggested in U.S. Pat. No.6,235,724.

It will be understood that these groups must still qualify as stronglylipophilic groups, which may further constrain the parameters indicatedabove.

The lipid structures depicted in our FIG. 4 are of particular interest.All of them qualify as strongly lipophilic groups.

Lipid component of Other Carbohydrate Ligand Analogs

The lipid A analogs of the present invention are required to comprise atleast one strongly lipophilic group. The carbohydrate ligand analogs ofthe present invention which are not lipid A analogs may, but need not,comprise a strongly lipophilic group. This can facilitate integrationinto a liposome. It should be noted that, for the purpose of determiningwhether an analog comprises a strongly lipophilic group, the requiredPet core is disregarded.

Definition of Lipophilic and Strongly Lipophilic Groups

Groups may be classified as lipophilic (hydrophobic), lipophobic(hydrophilic}, or neutral. The lipophilicity of groups may be determinedby measuring the partition coefficient of the molecule HZ (where Z isthe side chain in question) between a nonpolar solvent (e.g., ethanol,dioxane, acetone, benzene, n-octanol) and water, at STP. Thelipophilicity may be defined as the logarithm of this partitioncoefficient; it will then be positive for molecules which prefer thenonpolar solvent. Thus, a lipophilic group is one for which log P isgreater than zero.

The partition coefficient (P) is defined as the ratio of the equilibriumconcentrations of a dissolved substance in a two-phase system consistingof two largely immiscible solvents. One such system is n-octanol:water;the octanol phase will contain about 20% water and the water phase about0.008% octanol. Thus, the relevant partition coefficient (Pow) is theratio of the molar concentration of the solute in octanol saturated withwater to its molar concentration in water saturated with octanol.N-octanol is a useful surrogate for biological membranes because it,like many membrane components, is amphiphilic. (Reference hereafter tolog P shall mean log Pow, unless otherwise stated.)

For more information on methods of determining Pow, see Sangster, J.,Octanol-Water Partition Coefficients: Fundamentals and PhysicalChemistry (April 1997) (ISBN 0-471-9739).

For tabulations of octanol-water partition coefficients, see the EPA“Chemicals in the Environment: OPPT Chemicals Fact Sheets” the USDAPesticide Properties Database, Sangster, J., “Octanol-Water PartitionCoefficients of Simple Organic Compounds”, J. Phys. Chem. Ref. Data,18:1111-1230 (1989); Verbruggen, E. M. J., et al., “PhysiochemicalProperties of Higher Nonaromatic Hydrocarbons: Literature Study,” J.Phys. Chem. Ref. Data, 29:1435-46 (2000). For more sources, seereferences cited at Penn State University Libraries, Physical SciencesLibrary, octanol-water Partition Coefficients (last updated Aug. 21,2001), at the URL libraries.psu.edu/crsweb/physci/coefficients.htm. Itshould be noted that the Pow values compiled for different compounds mayhave been determined by different methodologies.

To avoid the need for experimental determinations of log Pow, for thepurpose of the present invention, the value predicted by Meylan's methodwill be used.

In Meylan's method, the predicted log Pow is obtained by adding weightedcoefficients for each fragment (the raw coefficient multiplied by thenumber of copies of that fragment) to the constant 0.2290. The fragmentsconsidered include aliphatically attached —CH3 (0.5473), —CH2— (0.4911),—CH (0.3614), —OH (−1.4086), —NH2 (−1.4148), —C(═O)N (−0.5236), —SH(−0.0001), —NH— (−1.4962), —N═C (−0.0010), —O— (−1.2566), —CHO(−0.9422), -tert C so 3+C attached (0.2676), C no H not tert (0.9723),—C(═O)O— (−0.9505), —C(═O)— (−1.5586), ═CH or C<(0.3836), #C (0.1334),—C(═O)N (−0.5236), —O—CO—C—N—CO (−0.5), —SO—O (−9), —O—P (−0.0162); O═P(−2.4239), phosphate attached —OH (0.475); aromatic C (0.2940), aromaticN (5 membered ring) (−0.5262), and aromatically attached —OH (−0.4802)

The Meylan algorithm is implemented in the program LogPow (KowWin). Anonline version of the program, available atesc.syrres.com/interkow/kowdemo.htm accepts either CAS registry numbersor SMILES structure notations. The program also reports experimentallydetermined values, if in its database.

A group is expected to be a lipophilic group if its log P, as predictedby the Meylan algorithm, is greater than zero.

For the purpose of this disclosure, a strongly lipophilic group isdefined as being a group, comprising at least five atoms other thanhydrogen, for which the predicted log P is at least 3.

Preferably, the log P predicted by the Meylan algorithm is at at least4, at least 5, at least 6, at least 7, at least 8, at least 9, or atleast 10, the higher the more preferred.

Preferably, the strongly lipophilic group will comprise not more than100 atoms other than hydrogen, more preferably, not more than 80 suchatoms, still more preferably, not more than 60 such atoms, even morepreferably not more than 40 such atoms.

As noted previously, the strongly lipophilic group must comprise atleast five atoms other than hydrogen. Preferably, it comprises at leastsix, more preferably at least 8, still more preferably at least 9, evenpreferably, it comprises at least 11 such atoms, still more preferablyat least 13 such atoms, most preferably at least 21 such atoms.

Preferably, the strongly lipophilic group has an elemental compositionlimited to the elements carbon, silicon, hydrogen, oxygen, nitrogen,sulfur, and phosphorous. Preferably, the majority of the bonds withinthe side chain which do not involve hydrogen are carbon-carbon bonds.

Since the presence of oxygen, nitrogen, sulfur and phosphorous tends toreduce lipophilicity, in the strongly lipophilic group, preferably morethan 50%, still more preferably more than 75%, of the non-hydrogen atomsare carbon atoms.

For the same reason, the strongly lipophilic group preferably comprisesat least 5, at least 6, at least 7, at least 8, at least 9, or at least10 carbon atoms.

Application of Definition of Lipophilicity

Using the program LogKow, we have calculated (see below) low Pow valuesfor the structures set forth in FIG. 4, or otherwise deemed worthy ofcomparison.

SMILES (lower case is arom) Comments PredLogP CCCCC alkyl (C5) 2.80CCCCC C alkyl (C6) 3.29 CCCCC CCCCC CCCCC CCCCC alkyl (C20) 10.16 CCCC OCCCC 3.01 CC(C) (C)C Pet Core 2.69 FIG. 4 structures CCCCC CCCCC CCCCalkyl (C14) 7.22 O═C CCCCC CCCCC CCC acyl (14:0) 5.73 CO CC(0) CCCCCCCCCC C 14:0, 3-OH 4.19 O═C CC(═O) CCCCC CCCCC 3.68 O═C CC(O C(═O)CCCCCCCCCC CCC) 14:0 3-O— 11.09 CCCCC CCCCC C (14:0) O═C CC(O C(═O)CCCCCC═CCCC CCC) 14:0 3-O— 10.87 CCCCC CCCCC C (14:1) O═C C(COC(═O)CCCCCCCCCC CCC) 11.61 CO C(═O) CCCCC CCCCC CCC O═C CC(NC(═O)CCCCC CCCCC CCC)N-linked 9.57 C(═O)O CCCCC CCCCC C secondary acyl O—C CC(OC(═O)CC(OCCCCC CCCCC has O-linked 15.65 CC) CCCCC CCCCC C)CCCCC CCCCC C tertiaryacyl chainThe predicted log P is used even if an experimental log P is available,e.g., for Pet core, it is 3.11.

Reference Carbohydrate Ligands; Carbohydrate Ligand Analogs

A reference carbohydrate ligand, for the purpose of the presentinvention, is a compound comprising one or more amino sugar units ashereafter defined, and which does not comprise a Pet core, which iscapable of binding specifically to a receptor as a result, at least inpart, of the presence of said sugar units. This reference ligand may,but need not, occur in nature.

The receptor may be a cellular receptor, or it may be an antibody. Theantibody may, but need not, be naturally occurring, e.g., as part of theimmune response to a disease. When the receptor is an antibody, theligand may be considered an antigen. If it is able to elicit an immuneresponse on its own, it is considered an immunogen. Otherwise, it isconsidered a hapten.

The reference carbohydrate ligand preferably has a specific bindingactivity for such receptor (desirably, with a binding affinitycharacterized by a K_(d) less—i.e., better-than 10-3 liters/mole) and,more preferably, a biological or immunological activity attributable tosuch receptor binding.

Some reference carbohydrate ligands are set forth in the section“Carbohydrate Haptens” below, and others are in FIGS. 19 and 20.

In addition, one may consider antibiotics which contain carbohydrate,such as the pure sugar nojirimycin, the aminoglycosides streptomycin,kanamycin and gentamycin C, the N-glycoside streptothricin, theC-glycoside vancomycin, and the glycolipid moenomycin A.

It may also be an antitumor ligand, such as various sulfatedoligosaccharides, in particular phosphomannopentaose sulfate (PI-88) andmaltohexaose sulfate. See Parish, et al., Cancer Res., 59: 3433-41(1999).

Or it may be an antiviral ligand, such as the azasugar 6-O-benzoylcastanospermine, an anti-Parkinson's disease agent, such as glycolipidganglioside G, an anti-convulsant, such as topiramate, or a glycosidaseinhibitor for diabetes therapy, such as an aza sugar, or ananti-thrombotic, such as the glycosylaminoglycan heparin.

The carbohydrate ligand analogs of the present invention are compoundswhich can compete with a reference carbohydrate ligand, as definedabove, for binding to a receptor, and which differ from the referencecarbohydrate ligand in that at least one amino sugar unit of thereference carbohydrate ligand is replaced with a (Pet core)-NH— moiety.They usually will be substantially identical to the referencecarbohydrate ligand, disregarding such replacement.

The reference carbohydrate ligand may comprise sugar units which are notamino sugars. It may also comprise substantial non-carbohydratemoieties, such as, without limitation, lipids, sulfates, phosphates,amino acids, and nucleobases. It thus may be a glycolipid orglycopeptide.

A carbohydrate ligand analog may be considered substantially identicalto the reference carbohydrate ligand if:

(1) for each sugar unit in the reference ligand, there is either acorresponding, substantially identical sugar unit or a corresponding Petunit in the analog.(2) The basic topology of the sugar units of the reference ligand issubstantially identical to that of the corresponding sugar or Pet unitsin the analog.

One sugar unit is considered substantially identical to another if

(1) they are both open or both cyclic,(2) if both cyclic, the ring sizes are the same and the ring heteroatomsare the same (usually oxygen),(3) if the configuration of a ring hydroxyl is constrained (axial orequatorial) in the reference ligand sugar, the hydroxyl is eitherretained in the analog sugar unit, or is replaced with halogen or withthiol,(4) if the constrained configuration hydroxyl is retained in the analogsugar unit, it is constrained the same way (axial or equatorial) in theanalog sugar unit,(5) ring carbons which are aminated in the reference ligand sugar unitare aminated in the analog sugar unit, and no other ring carbons areaminated;(6) the configuration (alpha or beta) of the anomeric carbon in thereference ligand sugar unit is retained in the analog sugar unit.

Permissible modifications include (1) replacement or deletion ofsubstituents, other than hydroxyl, on ring carbons of the referenceligand sugar unit, (2) replacement or deletion of substituents on thering carbons immediately adjacent to the ring heteroatom. Replacementcan be with a larger chemical moiety than the original moiety.

By way of example, galactose, glucose and fucose are all hexoses (6carbon sugar units), aldoses and pyranoses (with 6 membered rings; oneoxygen, five carbon atoms). They differ in that Gal has an axial 4-OH,Glc has an equatorial 4-OH, and Fuc has an axial 4-OH but is missing a6-OH, i.e., it is 6-deoxy-L-galactose. The carbons immediately adjacentto the ring oxygen are the C-1 and C-5 carbons. The C-1 substituent isOH, and the C-5 substituent is CH₂OH in Gal and Glc, and CH₃ in Fuc.

These C-1 and C-5 substituents can be freely deleted or replaced, exceptthat they cannot be aminated directly. The C-2, C-3 and C-4 atoms eachbear configuration-constrained hydroxyls. These can be replaced onlywith thiol or halogen.

The replacement or deletion of substituents is further limited if thesubstituent of the ring carbon of the reference ligand sugar unitcomprises another sugar unit. The substituent then cannot be deletedaltogether, and it can be replaced only by a substituent which comprisesa sugar unit or a Pet unit.

The basic topology is substantially identical if for each pair of sugarunits which are linked directly in the reference ligand, thecorresponding sugar units (or Pet units) must be linked directly in theanalog. Linkages are considered direct if they do not comprise anothersugar unit or Pet unit and if the most direct chain of atoms between thetwo units is not more than three times the length of the originallinkage. It is not necessary that the chemical nature of the linkage bethe same, e.g., a glycosidic linkage can be replaced by an etherlinkage.

By way of example, in an analog of Lewis X, there is only one aminosugar (GlcNAc), so it is replaced with Pet-NH—. There was also a Fucalpha-O-linked 1->4 to the amino sugar, and a Gal beta-O-linked 1->3 tothe same sugar. The analog would be Fuc alpha, linked through its C-1carbon to a moiety comprising Pet, the latter being linked to the C-1carbon of Gal beta. In both retained sugars, the C-5 substituent couldbe replaced or even eliminated (the sugars would then be pentoses ratherthan hexoses). Additionally, any of the C-2,C-3 and C-4 hydroxyls couldbe replaced with thiol or halogen.

It should be noted that the Lewis-X analogs would also be Lewis-aanalogs.

Pharmaceutically Acceptable Salts

The ligand analogs of the present invention also includepharmaceutically acceptable salts of the disclosed compounds.Pharmaceutically acceptable salts include, but are not limited to,sodium, potassium, calcium and magnesium salts.

Carbohydrate

The term “carbohydrate” (sugar) includes monosaccharides,oligosaccharides and polysaccharides, as well as substances derived fromthe monosaccharides by reduction of the carbonyl group (alditols), byoxidation of one or more terminal groups to carboxylic acids, or byreplacement of one or more hydroxy groups by a hydrogen atom, an aminogroup, a thiol group, or similar heteroatomic groups. It also includederivatives of the foregoing.

Monosaccharides

Parent monosaccharides are polyhydroxy aldehydes (H[CHOH]_(n)—CHO) orpolyhydroxy ketones (H—[CHOH]_(n)—CO—[CHOH]_(m)—H) with three or morecarbon atoms. The term “monosaccharide unit”, “carbohydrate unit” or“sugar unit” refers to a residue of a monosaccharide, including thederivatives of monosaccharides contemplated herein.

Each monosaccharide unit is preferably a triose (e.g., glyceraldehyde),tetrose (e.g., erythrose, threose), pentose (e.g., ribose, arabinose,xylose, lyxose), hexose (e.g., allose, altrose, glucose, mannose,gulose, idose, galactose, talose), heptose, or octose. More preferablyit is a pentose or hexose.

Each monosaccharide unit may be an aldose (having an aldehydic carbonylor potential aldehydic carbonyl group) or a ketose (having a ketoniccarbonyl or potential ketonic carbonyl group). (Fructose is an exampleof a ketose.) The monosaccharide unit further may have more than onecarbonyl (or potential carbonyl) group, and hence may be a dialdose,diketose, or aldoketose. The term “potential aldehydic carbonyl group”refers to the hemiacetal group arising from ring closure, and theketonic counterpart (the hemiketal structure).

The monosaccharide unit may be a cyclic hemiacetal or hemiketal. Cyclicforms with a three membered ring are oxiroses; with four, oxetoses, withfive, furanoses; with six, pyranoses; with seven, septanoses, witheight, octaviruses, and so forth. The locants of the positions of ringclosure may vary. Note that in the more common cyclic sugars, the ringconsists of one ring oxygen, the remaining ring atoms being carbon;hence, in pyranose, there is one ring oxygen and five ring carbons.

The monosaccharide unit may further be a deoxy sugar (alcoholic hydroxygroup replaced by hydrogen), amino sugar (alcoholic hydroxy groupreplaced by amino group), a thio sugar (alcoholic hydroxy group replacedby thiol, or C═O replaced by C═S, or a ring oxygen of cyclic formreplaced by sulfur), a seleno sugar, a telluro sugar, an aza sugar (ringcarbon replaced by nitrogen), an imino sugar (ring oxygen replaced bynitrogen), a phosphano sugar (ring oxygen replaced with phosphorus), aphospha sugar (ring carbon replaced with phosphorus), a C-substitutedmonosaccharide (hydrogen at a non-terminal carbon atom replaced withcarbon), an unsaturated monosaccharide, an alditol (carbonyl groupreplaced with CHOH group), aldonic acid (aldehydic group replaced bycarboxy group), a ketoaldonic acid, a uronic acid, an aldaric acid, andso forth. Amino sugars include glycosylamines, in which the hemiacetalhydroxy group is replaced.

Derivatives of these structures include O-substituted derivatives, inwhich the alcoholic hydroxy hydrogen is replaced by something else.Possible replacements include alkyl, acyl, phosphate, phosphonate,phosphinate, and sulfate. Likewise, derivatives of amino sugars includeN-substituted derivatives, and derivatives of thio sugars includeS-substituted derivatives.

Sialic acid, also known as N-acetyl neuraminic acid (NANA), is ofparticular interest. It is the terminal sugar on severaltumor-associated carbohydrate epitopes.

Combinations

Any of the carbohydrate ligand analogs of the present invention may beused in combination with each other, with other carbohydrate ligands(including, but not limited to, the reference carbohydrate ligands andto other analogs thereof), and other pharmaceutical agents. When theligand analog is used as an immunological agent, it may be used incombination with other immunological agents. Immunological agentsinclude antigens (including both immunogens and haptens), adjuvants, andother immodulatory molecules (including cytokines).

Any of the lipid A analogs of the present invention may be used incombination with each other, with other lipid A analogs, with naturallipid A molecules, and other pharmaceutical agents. The latter may beimmunological agents.

A combination may be a covalent conjugate, a noncovalent conjugate, asimple mixture, or use such that all of the elements of the combinationare simultaneously active in the subject to which they are administered.Simultaneous activity may, but need not, be achieved by simultaneousadministration. Compounds may be simultaneously active even if they arenot simultaneously administered, e.g., compound A with a long half-lifeis administered prior to compound B with a short half-life, but A isstill present in the body at an effective level when B is administered.

Immunogen

The immunogen of the present invention is a molecule, comprising atleast one disease-associated B or T cell epitope, as defined below, andwhich, when suitably administered to a subject (which, in some cases,may mean associated with a liposome or with an antigen-presenting cell),elicits a humoral and/or cellular immune response which is protectiveagainst the disease.

The present invention contemplates

-   -   (1) the use of the disclosed lipid A analogs to stimulate innate        immunity,    -   (2) the use of the disclosed lipid A analogs to adjuvant the        specific immune response to an administered immunogen, and    -   (3) the use of an immunogen comprising at least one of disclosed        carbohydrate ligand analogs to elicit a specific immune        response, with or without the use of the disclosed lipid A/Pet        analogs as adjuvants. (In case (3), the carbohydrate ligand        analog comprises a disease-associated carbohydrate epitope as        hereafter defined.)

If the epitope is a carbohydrate epitope, it may be an analog of anaturally occurring epitope containing at least one amino sugar, inwhich at least one amino sugar is replaced with an aminated Pet unit.

Epitope

The epitopes of the present invention may be B-cell or T-cell epitopes,and they may be of any chemical nature, including without limitationpeptides, carbohydrates, lipids, glycopeptides and glycolipids. Theepitope may be identical to a naturally occurring epitope, or a modifiedform of a naturally occurring, epitope.

A term such as “MUC1 epitope”, without further qualification, isintended to encompass, not only a native epitope of MUC1, but also amutant epitope which is substantially identical to a native epitope.Such a mutant epitope must be cross-reactive with a native MUC1 epitope.Likewise, a term such as “tumor-associated epitope” includes both nativeand mutant epitopes, but the mutant epitope must be cross-reactive witha native tumor-associated epitope.

B-Cell Epitopes

B-cell epitopes are epitopes recognized by B-cells and by antibodies.B-cell peptide epitopes are typically at least five amino acids, moreoften at least six amino acids, still more often at least seven or eightamino acids in length, and may be continuous (“linear”) or discontinuous(“conformational”) (the latter being formed by the folding of a proteinto bring noncontiguous parts of the primary amino acid sequence intophysical proximity). B-cell epitopes may also be carbohydrate epitopes.

T-Cell Epitopes

The T cell epitope, if any, may be any T cell epitope which is at leastsubstantially the same as a T-cell epitope of an antigen including ahapten) which is associated with a disease or adverse condition to adegree such that it could be prophylactically or therapeutically usefulto stimulate or enhance a cellular immune response to that epitope. Suchdiseases and conditions include, but are not limited to parasiticdiseases such as schistosomiasis and leishmania, fungal infections suchas candidiasis, bacterial infections such as leprosy, viral infectionssuch as HIV infections, and cancers, especially solid tumors. Of course,the greater the degree of specificity of the epitope for the associateddisease or adverse condition, the more likely it is that the stimulationof an immune response to that epitope will be free of adverse effects.

The epitope must, of course, be one amenable to recognition by T-cellreceptors so that a cellular immune response can occur. For peptides,the T-cell epitopes may interact with class I or class II MHC molecules.The class I epitopes usually 8 to 15, more often 9-11 amino acids inlength. The class II epitopes are usually 5-24 (a 24 mer is the longestpeptide which can fit in the Class II groove), more often 8-24 aminoacids. If the immunogen is larger than these sizes, it will be processedby the immune system into fragments of a size more suitable forinteraction with MHC class I or II molecules.

The carbohydrate T-cell epitopes may be as small as a single sugar unit(e.g., Tn). They are preferably no larger than five sugars.

Many T-cell epitopes are known. Several techniques of identifyingadditional T-cell epitopes are recognized by the art. In general, theseinvolve preparing a molecule which potentially provides a T-cell epitopeand characterizing the immune response to that molecule. Methods ofcharacterizing the immune response are discussed in a later section.

The reference to a CTL epitope as being “restricted” by a particularallele of MHC Class I molecules, such as HLA-A1, indicates that suchepitope is bound and presented by the allelic form in question. It doesnot mean that said epitope might not also be bound and presented by adifferent allelic form of MHC, such as HLA-A2, HLA-A3, HLA-B7, orHLA-B44.

Disease-Associated and Disease-Specific Epitopes

A disease is an adverse clinical condition caused by infection orparasitization by a virus, unicellular organism, or multicellularorganism, or by the development or proliferation of cancer (tumor)cells.

The unicellular organism may be any unicellular pathogen or parasite,including a bacteria, fungus or protozoan. The multicellular organismmay be any pathogen or parasite, including a protozoan, worm, orarthropod. Multicellular organisms include both endoparasites andectoparasites. Endoparasites are more likely to elicit an immuneresponse, but, to the extent they can elicit a protective immuneresponse, ectoparasites and their antigens are within the purview of thepresent invention.

An epitope may be said to be directly associated with a viral disease ifit is presented by a virus particle, or if it is encoded by the viralgenome and expressed in an infected cell.

An epitope may be said to be directly associated with a disease causedby a unicellular or multicellular organism if it presented by anintracellular, surface, or secreted antigen of the causative organism.

An epitope may be said to be directly associated with a particular tumorif it is presented by an intracellular, surface or secreted antigen ofsaid tumor. It need not be presented by all cell lines of the tumor typein question, or by all cells of a particular tumor, or throughout theentire life of the tumor. It need not be specific to the tumor inquestion. An epitope may be said to be “tumor associated” in general ifit is so associated with any tumor (cancer, neoplasm).

Tumors may be of mesenchymal or epithelial origin. Cancers includecancers of the colon, rectum, cervix, breast, lung, stomach, uterus,skin, mouth, tung, lips, larynx, kidney, bladder, prostate, brain, andblood cells.

An epitope may be indirectly associated with a disease if the epitope isof an antigen which is specifically produced or overproduced by infectedcells of the subject, or which is specifically produced or overproducedby other cells of the subject in specific, but non-immunological,response to the disease, e.g., an angiogenic factor which isoverexpressed by nearby cells as a result of regulatory substancessecreted by a tumor.

The term “disease associated epitope” also includes any non-naturallyoccurring epitope which is sufficiently similar to an epitope naturallyassociated with the disease in question so that antibodies or T cellswhich recognize the natural disease epitope also recognize the similarnon-natural epitope. Similar comments apply to epitopes associated withparticular diseases or classes of diseases.

An epitope may be said to be specific to a particular source (such as adisease-causing organism, or, more particular, a tumor), if it isassociated more frequently with that source than with other sources, toa detectable and clinically useful extent. Absolute specificity is notrequired, provided that a useful prophylactic, therapeutic or diagnosticeffect is still obtained.

In the case of a “specific tumor-specific” epitope, the epitope is morefrequently associated with that tumor that with other tumors, or withnormal cells. Preferably, there should be a statistically significant.(p=0.05) difference between its frequency of occurrence in associationwith the tumor in question, and its frequency of occurrence inassociation with (a) normal cells of the type from which the tumor isderived, and (b) at least one other type of tumor. An epitope may besaid to be “tumor-specific” in general is it is associated morefrequently with tumors (of any or all types) than with normal cells. Itneed not be associated with all tumors.

The term “tumor specific epitope” also includes any non-naturallyoccurring epitope which is sufficiently similar to a naturally occurringepitope specific to the tumor in question (or as appropriate, specificto tumors in general) so that antibodies or T cells stimulated by thesimilar epitope will be essentially as specific as CTLs stimulated bythe natural epitope.

In general, tumor-versus-normal specificity is more important thantumor-versus-tumor specificity as (depending on the route ofadministration and the particular normal tissue affected), higherspecificity generally leads to fewer adverse effects. Tumor-versus-tumorspecificity is more important in diagnostic as opposed to therapeuticuses.

The term “specific” is not intended to connote absolute specificity,merely a clinically useful difference in probability of occurrence inassociation with a pathogen or tumor rather than in a matched normalsubject.

In one embodiment, the epitope is a parasite-associated epitope, such asan epitope associated with leishmania, malaria, trypanosomiasis,babesiosis, or schistosomiasis. In another embodiment, the epitope is aviral epitope, such as an epitope associated with human immunodeficiencyvirus (HIV), Epstein-Barr virus (EBV), or hepatitis.

The epitope may also be associated with a bacterial antigen, such as anantigen of the tuberculosis bacterium, Staphylococcus, E. coli orShigella sonnei.

In another embodiment, the epitope is associated with a cancer (tumor),including but not limited to cancers of the respiratory system (lung,trachea, larynx), digestive system (mouth, throat, stomach, intestines)excretory system (kidney, bladder, colon, rectum), nervous system(brain), reproductive system (ovary, uterus, cervix), glandular system(breast, liver, pancreas, prostate), skin, etc. The two main groups ofcancers are sarcomas, which are of mesenchymal origin and affect suchtissues as bones end muscles, and carcinomas, which are of epithelialorigin and make up the great majority of the glandular cancers ofbreasts, stomach, uterus, skin and tongue. The sarcomas includefibrosarcomas, lymphosarcomas, osteosarcomas, chondrosarcomas,rhabdosarcomas and liposarcomas. The carcinomas include adenocarcinomas,basal cell carcinomas and squamous carcinomas.

Cancer-associated epitopes include, but are not limited to, peptideepitopes such as those of mutant p53, the point mutated Ras oncogenegene product, her 2/neu, c/erb2, and the MUC1 core protein, andcarbohydrate epitopes such as sialyl Tn (STn), TF, Tn, CA 125, sialylLe^(x), sialyl Le^(a) and P97.

Identification of Natural Epitopes

Naturally occurring epitopes may be identified by a divide-and-testprocess. One starts with a protein known to be antigenic or immunogenic.One next tests fragments of the protein for immunological activity.These fragments may be obtained by treatment of the protein with aproteolytic agent, or, if the peptide sequence is known, one maysynthetically prepare smaller peptides corresponding to subsequences ofthe protein. The tested fragments may span the entire protein sequence,or just a portion thereof, and they may be abutting, overlapping, orseparated.

If any of the fragments are immunologically active, the active fragmentsmay themselves be subjected to a divide-and-test analysis, and theprocess may be continued until the minimal length immunologically activesequences are identified. This approach may be used to identify eitherB-cell or T-cell epitopes, although the assays will of course bedifferent. Geysen teaches systematically screening all possibleoligopeptide (pref. 6-10 a.a.) abutting or overlapping fragments of aparticular protein for immunological activity in order to identifylinear epitopes. See WO 84/03564.

It is also possible to predict the location of B-cell or T-cell peptideepitopes if an amino acid sequence is available. B-cell epitopes tend tobe in regions of high local average hydrophilicity. See Hopp and Wood,Proc. Nat. Acad. Sci. (USA) 78: 3824 (1981); Jameson and Wolf, CABIOS,4: 181 (1988). T-cell epitopes can be predicted on the basis of knownconsensus sequences for the peptides bound to MHC class I molecules ofcells of a particular haplotype. See e.g., Slingluff, WO98/33810,especially pp. 15-16; Parker, et al., “Scheme for ranking potentialHLA-A2 binding peptides based on independent binding of individualpeptide side chains”, J. Immunol. 152: 163 (1994).

Naturally occurring T-cell epitopes may be recovered by dissociatingthem from their complexes with MHC class 1 molecules and then sequencingthem, e.g., by mass spectroscopic techniques.

Generally speaking, in addition to epitopes which are identical to thenaturally occurring disease- or tumor-specific epitopes, the presentinvention embraces epitopes which are different from but substantiallyidentical with such epitopes, and therefore disease- or tumor-specificin their own right. It also includes epitopes which are not substantialidentical to a naturally occurring epitope, but which are nonethelesscross-reactive with the latter as a result of a similarity in 3Dconformation.

Peptide Epitopes

A peptide epitope is considered substantially identical to a referencepeptide epitope (e.g., a naturally occurring epitope) if it has at least10% of an immunological activity of the reference epitope and differsfrom the reference epitope by no more than one non-conservativesubstitution.

Carbohydrate Haptens; Epitopes

The carbohydrate hapten of the present invention is a carbohydrate whichcomprises (and preferably is identical to) a carbohydrate epitope, butwhich does not elicit a humoral immune response by itself.

Normally, a carbohydrate hapten will not be a polysaccharide, as apolysaccharide is usually large enough to be immunogenic in its ownright. The borderline between an oligosaccharide and a polysaccharide isnot fixed, however, we will define an oligosaccharide as consisting of 2to 20 monosaccharide (sugar) units.

The hapten may be a monosaccharide (without glyosidic connection toanother such unit) or an oligosaccharide. If an oligosaccharide, itpreferably is not more than 10 sugar units.

Tumor associated carbohydrate epitopes are of particular interest.

A variety of carbohydrates can be conjugated according to the presentinvention, for use particularly in detecting and treating tumors. TheTn, T, sialyl Tn and sialyl (2->6)T haptens are particularly preferred.

In particular, for detecting and treating tumors, the three types oftumor-associated carbohydrate epitopes which are highly expressed incommon human cancers are conjugated to aminated compounds. Theseparticularly include the lacto series type 1 and type 2 chain, cancerassociated ganglio chains, and neutral glycosphingolipids.

Examples of the lacto series Type 1 and Type 2 chains are as follows:Lewis a, dimeric Lewis a, Lewis b, Lewis b/Lewis a, Lewis x, Lewis, y,Lewis a/Lewis x. dimeric Lewis x, Lewis y/Lewis x, trifucosyl Lewis y,trifucosyl Lewis b, sialosyl Lewis x, sialosyl Lewis y, sialosyl dimericLewis x, Tn, sialosyl Tn, sialosyl TF, TF. Examples of cancer-associatedganglio chains are as follows: GM3. GD3, GM2, GM4, GD2, GM1, GD-1a,GD-1b. Neutral sphingolipids include globotriose, globotetraose,globopentaose, isoglobotriose, isoglobotetraose, mucotriose,mucotetraose, lactotriose, lactotetraose, neolactotetraose,gangliotriose, gangliotetraose, galabiose, and 9-O-acetyl-GD3.

Numerous antigens of clinical significance bear carbohydratedeterminants. One group of such antigens comprises the tumor-associatedmucins (Roussel, et al., Biochimie 70, 1471, 1988).

Generally, mucins are glycoproteins found in saliva, gastric juices,etc., that form viscous solutions and act as lubricants or protectantson external and internal surfaces of the body. Mucins are typically ofhigh molecular weight (often >1,000,000 Dalton) and extensivelyglycosyiared. The glycan chains of mucins are O-linked (to serine orthreonine residues) and may amount to more than 80% of the molecularmass of the glycoprotein. Mucins are produced by ductal epithelial cellsand by tumors of the same origin, and may be secreted, or cell-bound asintegral membrane proteins (Burchell, et al., Cancer Res., 47, 5476,1987; Jerome, et al., Cancer Res., 51, 2908, 1991).

Cancerous tissues produce aberrant mucins which are known to berelatively less glycosylated than their normal counter parts (Hull, etal., Cancer Commun., 1, 261, 1989). Due to functional alterations of theprotein glycosylation machinery in cancer cells, tumor-associated mucinstypically contain short, incomplete glycans. Thus, while the normalmucin associated with human milk fat globules consists primarily of thetetrasaccharide glycan, gal β1-4 glcNAcp1-6(gal β1-3) gal NAc-α and itssialylated analogs (Hull, et al.), the tumor-associated Tn haptenconsists only of the monosaccharide residue,α-2-acetamido-3-deoxy-D-galactopyranosyl, and the T-hapten of thedisaccharideP-D-galactopyranosyl-(1-3)α-acetamido-2-deoxy-D-galactopyranosyl. Otherhaptens of tumor-associated mucins, such as the sialyl-Tn and thesialyl-(2-6)T haptens, arise from the attachment of terminal sialylresidues to the short Tn and T glycans (Hanisch, et al., Biol. Chem.Hoppe-Seyler, 370, 21, 1989; Hakormori, Adv. Cancer Res., 52:257, 1989;Torben, et al., Int. J. Cancer, 45 666, 1980; Samuel, et al., CancerRes., 50, 4801, 1990).

The T and Tn antigens (Springer, Science, 224, 1198, 1984) are found inimmunoreactive form on the external surface membranes of most primarycarcinoma cells and their metastases (>90% of all human carcinomas). Ascancer markers, T and Tn permit early immunohistochemical detection andprognostication of the invasiveness of some carcinomas (Springer).Recently, the presence of the sialyl-Tn hapten on tumor tissue has beenidentified as an unfavorable prognostic parameter (Itzkowitz, et al.Cancer, 66, 1960, 1990; Yonezawa, et al., Am. J. Clin. Pathol., 98 167,1992). Three different types of tumor-associated carbohydrate antigensare highly expressed in common human cancers. The T and Tn haptens areincluded in the lacto series type, and type 2 chains. Additionally,cancer-associated ganglio chains and glycosphingolipids are expressed ona variety of human cancers.

The altered glycan determinants displayed by the cancer associatedmucins are recognized as non-self or foreign by the patient's immunesystem (Springer). Indeed, in most patients, a strong autoimmuneresponse to the T hapten is observed. These responses can readily bemeasured, and they permit the detection of carcinomas with greatersensitivity and specificity, earlier than has previously been possible.Finally, the extent of expression of T and Tn often correlates with thedegree of differentiation of carcinomas. (Springer).

An extensive discussion of carbohydrate haptens appears in Wong, U.S.Pat. No. 6,013,779. A variety of carbohydrates can be incorporated intoa synthetic glycolipopeptide immunogen, according to the presentinvention, for use particularly in detecting and treating tumors. TheTn, T, sialyl Tn and sialyl (2-->6)T haptens are particularly preferred.In particular, for detecting and treating tumors, the three types oftumor-associated carbohydrate epitopes which are highly expressed incommon human cancers are conjugated to aminated compounds. Theseparticularly include the lacto series type 1 and type 2 chain, cancerassociated ganglio chains, and neutral glycosphingolipids.

Examples of the lacto series Type 1 and Type 2 chains are as follows:

Examples of cancer-associated ganglio chains that can be conjugated toaminated compounds according to the present invention are as follows:

In addition to the above, neutral glycosphingolipids can also beconjugated to aminated compounds according to the present invention:

Selected Neutral Glycosphingolipids Globotriose: Galα→4Galβ1→4Glcβ1→Globotetraose: GalNAcβ1→3Galα→4Galβ1→4Glcβ1→ Globopentaose:GalNAcα1→3GalNAcβ1→3Galα→4Galβ1→4Glcβ1→ Isoglobotriose:Galα→3Galβ1→4Glcβ1→ Isoglobotetraose: GalNAcβ1→3Galα1→3Galβ1→4Glcβ1→Mucotriose: Galβ1→4Galβ1→4Glcβ1→ Mucotetraose:Galβ1→3Galβ1→4Galβ1→4Glcβ1→ Lactotriose: GalNAcβ1→3Galβ1→4Glcβ1→Lactotetraose: GalNAcβ1→3GalNAcβ1→3Galβ1→4Glcβ1→ Neolactotetraose:Galβ1→4GlcNAcβ1→3Galβ1→4Glcβ1→ Gangliotriose: GalNAcβ1→4Galβ1→4Glcβ1→Gangliotetraose: Galβ1→GlcNAcβ1→4Galβ1→4Glcβ1→

Galαbiose: Galα→4Galβ1→

9-O-Acetyl-GD3: 9-O-Ac-NeuAcα2→8NeuAcα2→3Galβ1→4Glcβ1→ Immunoconjugates

The immunogen of the present invention may be an immunoconjugate inwhich one or more epitopes are joined with other chemical moieties tocreate a molecule with different immunological properties, such asincreased ability to elicit a humoral immune response. For example, oneor more epitopes may be conjugated to a macromolecular carrier, such asalbumin, keyhole limpet hemocyanin (KLH) or polydextran. Or severalepitopes may be joined to a branched lysine core, such as a MAP-4peptide. Or several epitopes may simply be conjugated together usingsome other linker or molecular scaffold.

Adjuvants

It is generally understood that a synthetic antigen of low molecularweight can be weakly immunogenic, which is the biggest obstacle to thesuccess of a fully synthetic vaccine. One way to improve theimmunogenicity of such a synthetic antigen is to deliver it in theenvironment of an adjuvant.

As conventionally known in the art, adjuvants are substances that act inconjunction with specific antigenic stimuli to enhance the specificresponse to the antigen. An ideal adjuvant is believed tonon-specifically stimulate the immune system of the host, which upon thesubsequent encounter of any foreign antigen can produce strong andspecific immune response to that foreign antigen. Such strong andspecific immune response, which is also characterized by its memory, canbe produced only when T-lymphocytes (T-cells) of the host immune systemare activated.

T-cell blastogenesis and IFN-gamma production are two importantparameters for measuring the immune response. Experimentally, T-cellblastogenesis measures DNA synthesis that directly relates to T-cellproliferation, which in turn is the direct result of the T-cellactivation. On the other hand, IFN-gamma is a major cytokine secreted byT-cells when they are activated. Therefore, both T-cell blastogenesisand IFN-gamma production indicate T-cell activation, which suggests theability of an adjuvant in helping the host immune system to induce astrong and specific immune response to any protein-based antigen.

The compound is considered an adjuvant if it significantly (p=0.05)increases the level of either T-cell blastogenesis or of interferongamma production in response to at least one liposome/immunogencombination relative to the level elicited by the immunogen alone.Preferably, it does both. Preferably, the increase is at least 10%, morepreferably at least 50%, still more preferably, at least 100%.

Preferably, the toxicity of the lipid compounds of the present inventionis not more. than 50% that of said natural Lipid-A product; morepreferably it is less than 10% that of the latter.

A large number of adjuvants are known in the art, including Freund'scomplete adjuvant, saponin, DETOX (Ribi Immunochemicals), MontanideISA-51, -50 and -70, QS-21, monophosphoryl lipid A and analogs thereof.A lipid adjuvant can be presented in the context of a liposome.

The present liposomal vaccines may be formulated advantageously with anadjuvant. Monophosphoryl lipid A (MPLA), for example, is an effectiveadjuvant that causes increased presentation of liposomal antigen tospecific T Lymphocytes. Alving, C. R., Immunobiol., 187:430-446, (1993).The skilled artisan will recognize that lipid-based adjuvants, such asLipid A and derivatives thereof, are also suitable. A muramyl dipeptide(MDP), when incorporated into liposomes, has also been shown to increaseadjuvanticity (Gupta R K et al., Adjuvants-A balance between toxicityand adjuvanticity,” Vaccine, 11, 293-306 (1993)).

Use of an adjuvant is not required for immunization.

Liposome Formulations

Liposomes are microscopic vesicles that consist of one or more lipidbilayers surrounding aqueous compartments. See e.g., Bakker-Woudenberget al., Eur. J. Clin. Microbiol. Infect. Dis. 12 (Supp1.1): S61 (1993)and Kim, Drugs, 46: 618 (1993). Because liposomes can be formulated withbulk lipid molecules that are also found in natural cellular membranes,liposomes generally can be administered safely and are biodegradable.

Liposomes are globular particles formed by the physical self-assembly ofpolar lipids, which define the membrane organization in liposomes.Liposomes may be formed as uni-lamellar or multi-lamellar vesicles ofvarious sizes. Such liposomes, though constituted of small moleculeshaving no immunogenic properties of their own, behave likemacromolecular particles and display strong immunogenic characteristics.

Depending on the method of preparation, liposomes may be unilamellar ormultilamellar, and can vary in size with diameters ranging from about0.02 microm to greater than about 10 microm. A variety of agents can beencapsulated in liposomes. Hydrophobic agents partition in the bilayersand hydrophilic agents partition within the inner aqueous space(s). Seee.g., Machy et al., Liposomes in Cell Biology and Pharmacology (JohnLibbey, 1987), and Ostro et al., American J. Hosp. Pharm. 46: 1576(1989).

Liposomes can adsorb to virtually any type of cell and then release anincorporated agent. Alternatively, the liposome can fuse with the targetcell, whereby the contents of the liposome empty into the target cell.Alternatively, a liposome may be endocytosed by cells that arephagocytic. Endocytosis is followed by intralysosomal degradation ofliposomal lipids and release of the encapsulated agents. Scherphof etal., Ann. N.Y. Acad. Sci., 446: 368 (1985).

Other suitable liposomes that are used in the methods of the inventioninclude multilamellar vesicles (MLV), oligolamellar vesicles (OLV),unilamellar vesicles (UV), small unilamellar vesicles (SUV),medium-sizedunilamellar vesicles (MUV), large unilamellar vesicles (LUV), giantunilamellar vesicles (GUV), multivesicular vesicles (MVV), single oroligolamellar vesicles made by reverse-phase evaporation method (REV),multilamellar vesicles made by the reverse-phase evaporation method(MLV-REV), stable plurilamellar vesicles (SPLV), frozen and thawed MLV(FATMLV), vesicles prepared by extrusion methods (VET), vesiclesprepared by French press (FPV), vesicles prepared by fusion (FUV),dehydration-rehydration vesicles (DRV), and bubblesomes (BSV). Theskilled artisan will recognize that the techniques for preparing theseliposomes are well known in the art. See Colloidal Drug DeliverySystems, vol. 66 (J. Kreuter, ed., Marcel Dekker, Inc., 1994).

A “liposomal formulation” is an in vitro-created lipid vesicle in whicha pharmaceutical agent, such as an antigen, of the present invention canbe incorporated or to which one can be attached. Thus,“liposomally-bound” refers to an agent that is partially incorporated inor attached to a liposome. The immunogen of the present invention may bea liposomally-bound antigen which, but for said liposome, would not bean immunogen, or it may be immunogenic even in a liposome-free state.Several different agents may be incorporated into or attached to thesame liposome, or different agents may be associated with differentliposomes, and the liposomes administered separately or together to asubject.

A lipid-containing molecule can be incorporated into a liposome becausethe lipid portion will spontaneously integrate into the lipid bilayer.Thus, a lipid-containing agent may be presented on the “surface” of aliposome. Alternatively, an agent may be encapsulated within a liposome.

Formation of a liposome requires one or more lipids. Any lipids may beused which, singly or in combination, can form a liposome bilayerstructure. Usually, these lipids will include at least one phospholipid.The phospholipids may be phospholipids from natural sources, modifiednatural phospholipids, semisynthetic phospholipids, fully syntheticphospholipids, or phospholipids (necessarily synthetic) with normaturalhead groups. The phospholipids of greatest interest are phosphatidylcholines, phosphatidyl phosphatidyl ethanolamines, phosphatidyl serines,phosphatidyl glycerols, phosphatidic acids, and phosphatidyl inositols.

The liposome may include neutral, positively charged, and/or negativelycharged lipids. Phosphatidyl choline is a neutral phospholipid.Phosphatidyl glycerol is a negatively charged glycolipid.N-[1-(2,3-dioleylox)propyl]-N,N,N-trimethylammonium chloride is apositively charged synthetic lipid. Another is3-beta-[N—(N′,N″-dimethylaminoethane)-carbamoyl]-cholesterol.

Usually, the lipids will comprise one or more fatty acid groups. Thesemay be saturated or unsaturated, and vary in carbon number, usually from12-24 carbons. The phospholipids of particular interest are those withthe following fatty acids: C12:0, C14:0, C16:0, C18:0, C18:1, C18:2,C18:3 (alpha and gamma) C20:0, C20:1, C20:3, C20:4, C20:5, C22:0, C22:5,C22:6, and C24:0, where the first number refers to the total number ofcarbons in the fatty acids chain, and the second to the number of doublebonds. Fatty acids from mammalian or plant sources all have even numbersof carbon atoms, and their unsaturations are spaced at three carbonintervals, each with an intervening methylene group.

Cholesterol reduces the permeability of “fluid-crystalline state”bilayers.

A liposome may include lipids with a special affinity for particulartarget cells. For example, lactosylceramide has a specific affinity forhepatocytes (and perhaps also for liver cancer cells).

In a preferred liposome formulation, the component lipids includephosphatidyl choline. More preferably they also include cholesterol, andstill more preferably, also phosphatidyl glycerol. Taking advantage ofthe self-assembling properties of lipids, one or more immunogens may beattached to the polar lipids that in turn become part of the liposomeparticle. Each immunogen comprises one or more antigenic determinants(epitopes). These epitopes may be B-cell epitopes (recognized byantibodies) or T-cell epitopes (recognized by T-cells). The liposome canact to adjuvant the immune response elicited by the associatedimmunogens. It is likely to be more effective than an adjuvant that issimply mixed with an immunogen, as it will have a higher local effectiveconcentration.

Moreover, a hapten may be attached in place of the aforementionedimmunogen. Like an immunogen, a hapten comprises an antigenicdeterminant, but by definition is too small to elicit an immune responseon its own (typically, haptens are smaller than 5,000 daltons). In thiscase, the lipid moiety may act, not only as an adjuvant, but also as animmunogenic carrier, the conjugate of the hapten and the lipid acting asa synthetic immunogen (that is, a substance against which humoral and/orcellular immune responses may be elicited).

Even if the lipid does not act as an immunogenic carrier, the liposomeborne hapten may still act as a synthetic antigen (that is, a substancewhich is recognized by a component of the humoral or cellular immunesystem, such as an antibody or T-cell). The term “antigen” includes bothhaptens and immunogens.

Thus, in some embodiments, the invention contemplates a liposome whosemembrane comprises a Lipid A analog as disclosed herein, and at leastone B-cell or T-cell epitope. The epitope may be furnished by alipopeptide, glycolipid or glycolipopeptide.

The lipidation of an immunogen normally will facilitate theincorporation of the immunogen into a liposome, which in turn canimprove the immune presentation of the immunogen. For most efficientincorporation, at least one strongly lipophilic group of the immunogenpreferably should be similar in size to at least one of the lipidcomponents of the liposome. For example, the size should be in the rangeof 50%-200% of the size of the reference lipid component of theliposome. Size may be measured by counting the number of non-hydrogenatoms of each, by calculating the molecular weight of each, or bycalculating (with the aid of 3D molecular models) the molecular volumeor longest dimension of each.

Preferably, the lipidated immunogen comprises a lipophilic moiety whichadjuvants the humoral or cellular immune response to the immunogen.

Unlike the bacterial adjuvant preparations, a synthetic Lipid-A analogcontributes a structurally well-defined lipid to the liposome membrane.Such defined structures not only reduce the burden of re-affirming the‘active’ membrane components after formulation, but also contribute tothe definition of liposome membrane. Such liposomes may be designated as‘totally synthetic vaccine formulations’ containing synthetic Lipid-Aanalog as an adjuvant and a synthetic lipid-containing antigen.

Characterizing the Immune Response

The cell-mediated immune response may be assayed in vitro or in vivo.The conventional in vitro assay is a T cell proliferation assay. A bloodsample is taken from an individual who suffers from the disease ofinterest, associated with that disease, or from a vaccinated individual.The T cells of this individual should therefore be primed to respond toa new exposure to that antigen by proliferating. Proliferation requiresthymidine because of its role in DNA replication.

Generally speaking, T cell proliferation is much more extensive than Bcell proliferation, and it may be possible to detect a strong T cellresponse in even an unseparated cell population. However, purificationof T cells is desirable to make it easier to detect a T cell response.Any method of purifying T cells which does not substantially adverselyaffect their antigen-specific proliferation may be employed. In ourpreferred procedure, whole lymphocyte populations would be firstobtained via collection (from blood, the spleen, or lymph nodes) onisopycnic gradients at a specific density of 10.7, ie Ficoll-Hypague orPercoll gradient separations. This mixed population of cells could thenbe further purified to a T cell population through a number of means.The simplest separation is based on the binding of B cell andmonocyte/macrophage populations to a nylon wool column. The T cellpopulation passes through the nylon wool and a >90% pure T populationcan be obtained in a single passage. Other methods involve the use ofspecific antibodies to B cell and or monocyte antigens in the presenceof complement proteins to lyse the non-T cell populations (negativeselection). Still another method is a positive selection technique inwhich an anti-T cell antibody (CD3) is bound to a solid phase matrix(such as magnetic beads) thereby attaching the T cells and allowing themto be separated (e.g., magnetically) from the non-T cell population.These may be recovered from the matrix by mechanical or chemicaldisruption.

Once a purified T cell population is obtained it is cultured in thepresence of irradiated antigen presenting cells (splenic macrophages, Bcells, dendritic cells all present). (These cells are irradiated toprevent them from responding and incorporating tritiated thymidine). Theviable T cells (100,000-400,000 per well in 100 μl media supplementedwith IL2 at 20 units) are then incubated with test peptides or otherantigens for a period of 3 to 7 days with test antigens atconcentrations from 1 to 100 pg/mL.

At the end of the antigen stimulation period a response may be measuredin several ways. First the cell free supernatants may be harvested andtested for the presence of specific cytokines. The presence ofa-interferon, IL2 or IL12 are indicative of a Th helper type 1population response. The presence of IL4, IL6 and IL10 are togetherindicative of a T helper type 2 immune response. Thus this method allowsfor the identification of the helper T cell subset.

A second method termed blastogenesis involves the adding tritiatedthymidine to the culture (e.g., 1 pcurie per well) at the end of theantigen stimulation period, and allowing the cells to incorporate theradiolabelled metabolite for 4-16 hours prior to harvesting on a filterfor scintillation counting. The level of radioactive thymidineincorporated is a measure of the T cell replication activities. Negativeantigens or no antigen control wells are used to calculated theblastogenic response in terms of a stimulation index. This is CPMtest/CPM control. Preferably the stimulation index achieved is at least2, more preferably at least 3, still more preferably 5, most preferablyat least 10.

CMI may also be assayed in vivo in a standard experimental animal, e.g.,a mouse. The mouse is immunized with a priming antigen. After waitingfor the T cells to respond, the mice are challenged by footpad injectionof the test antigen. The DTH response (swelling of the test mice iscompared with that of control mice injected with, e.g., saline solution.

Preferably, the response is at least 0.10 mm, more preferably at least0.15 mm, still more preferably at least 0.20 mm, most preferably atleast 0.30 mm.

The humoral immune response, in vivo, is measured by withdrawing bloodfrom immunized mice and assaying the blood for the presence ofantibodies which bind an antigen of interest. For example, test antigensmay be immobilized and incubated with the samples, thereby capturing thecognate antibodies, and the captured antibodies then measured byincubating the solid phase with labeled anti-isotypic antibodies.

Preferably, the humoral immune response, if desired, is at least asstrong as that represented by an antibody titer of at least 1/100, morepreferably at least 1/1000, still more preferably at least 1/10.000.

Lipid A analogs as immunostimulating agents

Lipid A analogs which have LPS/lipid A agonistic activities can be usedas immune stimulatory agents. They are potentially useful asimmunotherapeutic agents for the treatment of a wide range of diseases,e.g., infections and cancers. As demonstrated herein, these lipid Aanalogs are potent vaccine adjuvants. An immunostimulatory adjuvantstimulates the production of cytokines required for antigen specificantibody response, and cell-mediated immune responses including acytotoxic-lymphocytes, in the immunized host.

The compounds of the present invention can be formulated with apharmaceutically acceptable carrier for injection or ingestion. Thepharmaceutically acceptable carrier is a medium that does not interferewith the immunomodulatory activity of the active ingredient and is nottoxic to the host to which it is administered. Pharmaceuticallyacceptable carriers include without limitation oil-in-water orwater-in-oil emulsions, aqueous compositions, liposomes, micro beads andmicrosomes. As vaccine adjuvants, they can be formulated together withantigens to provide stronger immune responses and improve vaccineefficacy. Typically, an antigen is formulated in combination orseparately with an immunostimulatory adjuvant compounds such as thosedescribed in the present invention, to provide the pharmaceuticalcomposition. In other formulations, an antigen may be covalently linkedto an amino, carboxyl, hydroxyl, and/or phosphate moiety of the adjuvantcompounds of the present invention.

Antigens may be derived from pathogenic and non-pathogenic organisms,viruses, and fungi, or may be the whole organism. More specifically, theantigenic agent may be selected from the group consisting of: (1) live,heat killed, or chemically attenuated viruses, bacteria, mycoplasmas,fungi, and protozoa; (2) fragments, extracts, subunits, metabolites andrecombinant constructs of (1); (3) fragments, subunits, metabolites andrecombinant constructs of mammalian proteins and glycoproteins; (4)tumor-associated and tumor-specific antigens; and (5) nucleic acids.

The therapeutic composition may therefore utilize any suitable antigenor vaccine component in combination with an immunostimulating compoundof the present invention as an adjuvant. Such therapeutic compositionsmay suitably comprise proteins, peptides, glycopeptides and glycolipidswhich are pharmaceutically active for disease states and conditions suchas cancers, malaria, smallpox, anthrax, and SARS (sudden acuterespiratory syndrome).

The modes of administration may comprise the use of any suitable meansand/or methods for delivering the immunostimulatory adjuvant, adjuvantcontaining vaccine, or adjuvant and/or antigen to the host. Deliverymodes may include, but not limited to, parenteral administrationmethods, such as subcutaneous (SC) injection, transcutaneous, intranasal(IN), ophthalmic, transdermal, intramuscular (IM), intradermal (ID),intraperitoneal (IP), intravaginal, pulmonary, and rectaladministration, as well as non-parenteral, e.g. oral administration.

The immunostimulatory agents of the present invention may be usefullyadministered to the host with other therapeutic agents for the treatmentof targeted diseases in combined therapy to achieve better efficacy. Forexample, they can be used in combination with antibiotics, anti-viralagents, and anti-inflammatory agents to provide better treatment forinfections and autoimmune diseases. Formulation comprising of theimmunostimulatory compounds of the present invention can includeadditional components such as saline, oil, squalene, and otherimmunostimulatory compounds such as muramyl peptide analogs, bacterialDNA, CpG-oligonucleotide analogs, QS-21 (an immunostimulatory adjuvantderived from plant), and lipid A analogs not of the present inventiondisclosure.

Lipid A Analogs as Bacterial Endotoxin Antagonists

Lipid A analogs with LPS/lipid A antagonistic activity may be used forthe control of LPS-mediated pathophysiological disorders. UponGram-negative bacterial infection in humans, bacterial endotoxin,lipopolysaccharides (LPS), are released into the blood streams. Acuteinflammatory responses to LPS or its active principle lipid A result inthe release of cytokines and other cellular mediators, including tumornecrosis factor-α (TNF-α), interleukun-1 (IL-1), IL-6 and leukotrienesfrom monocytes and macrophages. At extreme levels, these cytokines andcellular mediators are known to trigger many pathophysiological eventsincluding fever, shock, hypotension, and organ failure (R. C. Bone,Clin. Microbiol. Rev. 1993, 6, 57). These events are generally termed asseptic syndrome. Sepsis is deadly and kills tens of thousands of peopleannually in US alone.

One strategy to control LPS-mediated disorders is to prevent LPS/lipid Abinding to receptors with inactive competitors (antagonists) ofLPS/lipid A. Lipid A analogs disclosed herein, due to their structuralsimilarity to the natural lipid A molecules, are expected to bind to theLPS-binding receptor, Toll-like receptor 4 (TLR4), but withouttriggering the un-controlled release of inflammatory cytokines by theimmune system. As LPS/lipid A-antagonists, such lipid A analogs caninhibit LPS-induced production of cytokines and thus confer benefits incontrolling LPS-mediated pathophysiological disorders.

As LPS-antagonists to neutralize the toxicity of bacterial endotoxin,such lipid A compositions are expected to display higher therapeuticbenefits when administered at early stage of bacterial infections. Inaddition, such lipid A analogs could be administered in conjunction withcommon antibiotics to relieve the burden to the host caused by theinfections. In short, the lipid A analogs described herein asLPS-antagonists are useful therapeutic agents for the treatment orprevention of LPS-mediated disorders resulting from Gram-negativebacterial infections. Such disorders include, without limitation, fever,generalized inflammation, disseminated intravascular coagulation,hypotension, acute renal failures, acute respiratory distress syndrome,hepatocellular destruction, and cardiac failure.

Another embodiment of the application of lipid A analogs disclosedherein is to suppress LPS-mediated virus production. LPS potentlystimulates the production of viruses which reside in monocytes ormacrophages (Ponerantz et al. J. Exp. Med. 1990, 127, 253). In the caseof HIV-1, increased viral production likely results from activation ofcells by both a direct activation by LPS and the LPS-mediated elevationin TNF-α levels. Cellular activation promotes increased binding oftrans-acting factors to the HIV-1 NF-KB binding site, which in turnleads to increased viral transcription and replication (Duh et al.;Proc. Natl. Acad. Sci. USA, 1989, 85, 5974). Thus, as LPS-antagonists,the lipid A analogs disclosed herein can inhibit an LPS-mediatedincrease in HIV-1 replication. Similarly, these lipid A analogs may beused to suppress the activation of any virus whose replication isdirectly or indirectly controlled by an NF-KB regulatory region. Suchviruses include, without limitation, cytomegalovirus or Herpes viruses.Furthermore, LPS has been implicated in influenza virus activation (Nainet al., J. Immunol. 1990, 145, 1921), and an enhanced release of TNF-αhas been suggested to be related with the observed complications ofcombined influenza and bacterial infections. Therefore the lipid Aanalogs with LPS-antagonistic activities disclosed herein may be used tosuppress influenza virus activation as well. In brief, the compositionsof the present invention can provide useful therapeutics for thetreatment or prevention of LPS-mediated exacerbation of latent or activeviral infections, e.g., infection with HIV-1, cytomegaloviruses, herpessimplex viruses, and influenza virus.

Pharmaceutical Subjects, Preparations and Methods

Applicants hereby incorporate by reference the discussion at pp. 32-46of WO98/33810.

Subjects

The recipients of the vaccines of the present invention may be anyvertebrate animal which can acquire specific immunity via a humoral orcellular immune response.

Among mammals, the preferred recipients are mammals of the OrdersPrimata (including humans, apes and monkeys), Arteriodactyla (includinghorses, goats, cows, sheep, pigs), Rodenta (including mice, rats,rabbits, and hamsters), and Carnivora (including cats, and dogs). Amongbirds, the preferred recipients are turkeys, chickens and other membersof the same order. The most preferred recipients are humans.

The preferred animal subject of the present invention is a primatemammal. By the term “mammal” is meant an individual belonging to theclass Mammalia, which, of course, includes humans. The invention isparticularly useful in the treatment of human subjects, although it isintended for veterinary uses as well. By the term “non-human primate” isintended any member of the suborder Anthropoidea except for the familyHominidae. Such non-human primates include the superfamily Ceboidea,family Cebidae (the New World monkeys including the capuchins, howlers,spider monkeys and squirrel monkeys) and family Callithricidae(including the marmosets); the superfamily Cercopithecoidea, familyCercopithecidae (including the macaques, mandrills, baboons, proboscismonkeys, mona monkeys, and the sacred hunaman monkeys of India); andsuperfamily Haminoidae, family Pongidae (including gibbons, orangutans,gorillas, and chimpanzees). The rhesus monkey is one member of themacaques.

Pharmaceutical Compositions

Pharmaceutical preparations of the present invention, comprise at leastone immunogen in an amount effective to elicit a protective immuneresponse. The response may be humoral, cellular, or a combinationthereof. The composition may comprise a plurality of immunogens.

At least one immunogen will be either a glycolipopeptide which isimmunogenic per se, or a glycolipopeptide which is immunogenic as aresult of its incorporation into a liposome.

The composition preferably further comprises a liposome. Preferredliposomes include those identified in Jiang, et al., PCT/US00/31281,filed Nov. 15, 2000 (our docket JIANG3A-PCT), and Longenecker, et al.,08/229,606, filed Apr. 12, 1994 (our docket LONGENECKER5—USA, andPCT/US95/04540, filed Apr. 12, 1995 (our docket LONGENECKER5—PCT).

The composition may comprise antigen-presenting cells, and in this casethe immunogen may be pulsed onto the cells, prior to administration, formore effective presentation.

The composition may contain auxiliary agents or excipients which areknown in the art. See, e.g., Berkow et al, eds., The Merck Manual, 15thedition, Merck and Co., Rahway, N.J., 1987; Goodman et al., eds.,Goodman and Gilman's The Pharmacological Basis of Therapeutics, 8thedition, Pergamon Press, Inc., Elmsford, N.Y., (1990); Avery's DrugTreatment: Principles and Practice of Clinical Pharmacology andTherapeutics, 3rd edition, ADIS Press, LTD., Williams and Wilkins,Baltimore, Md. (1987), Katzung, ed. Basic and Clinical Pharmacology,Fifth Edition, Appleton and Lange, Norwalk, Conn. (1992), whichreferences and references cited therein, are entirely incorporatedherein by reference.

A composition may further comprise an adjuvant to nonspecificallyenhance the immune response. Some adjuvants potentiate both humoral andcellular immune response, and other are specific to one or the other.Some will potentiate one and inhibit the other. The choice of adjuvantis therefore dependent on the immune response desired.

A composition may include immunomodulators, such as cytokines whichfavor or inhibit either a cellular or a humoral immune response, orinhibitory antibodies against such cytokines.

A pharmaceutical composition according to the present invention mayfurther comprise at least one cancer chemotherapeutic compound, such asone selected from the group consisting of an anti-metabolite, ableomycin peptide antibiotic, a podophyllin alkaloid, a Vinca alkaloid,an alkylating agent, an antibiotic, cisplatin, or a nitrosourea. Apharmaceutical composition according to the present invention mayfurther or additionally comprise at least one viral chemotherapeuticcompound selected from gamma globulin, amantadine, guanidine,hydroxybenzimidazole, interferon-α, interferon-β, interferon-γ,thiosemicarbarzones, methisazone, rifampin, ribvirin, a pyrimidineanalog, a purine analog, foscarnet, phosphonoacetic acid, acyclovir,dideoxynucleosides, or ganciclovir. See, e.g., Katzung, supra, and thereferences cited therein on pages 798-800 and 680-681, respectively,which references are herein entirely incorporated by reference.

Anti-parasitic agents include agents suitable for use againstarthropods, helminths (including roundworns, pinworms, threadworms,hookworms, tapeworms, whipworms, and Schistosomes), and protozoa(including amebae, and malarial, toxoplasmoid, and trichomonadorganisms). Examples include thiabenazole, various pyrethrins,praziquantel, niclosamide, mebendazole, chloroquine HCl, metronidazole,iodoquinol, pyrimethamine, mefloquine HCl, and hydroxychloroquine HCl.

Pharmaceutical Purposes

A purpose of the invention is to protect subjects against a disease. Theterm “protection”, as in “protection from infection or disease”, as usedherein, encompasses “prevention,” “suppression” or “treatment.”“Prevention” involves administration of a Pharmaceutical compositionprior to the induction of the disease. “Suppression” involvesadministration of the composition prior to the clinical appearance ofthe disease. “Treatment” involves administration of the protectivecomposition after the appearance of the disease. Treatment may beameliorative or curative.

It will be understood that in human and veterinary medicine, it is notalways possible to distinguish between “preventing” and “suppressing”since the ultimate inductive event or events may be unknown, latent, orthe patient is not ascertained until well after the occurrence of theevent or events. Therefore, it is common to use the term “prophylaxis”as distinct from “treatment” to encompass both “preventing” and“suppressing” as defined herein. The term “protection,” as used herein,is meant to include “prophylaxis.” See, e.g., Berker, supra, Goodman,supra, Avery, supra and Katzung, supra, which are entirely incorporatedherein by reference, including all references cited therein.

The “protection” provided need not be absolute, i.e., the disease neednot be totally prevented or eradicated, provided that there is astatistically significant improvement (p=0.05) relative to a controlpopulation. Protection may be limited to mitigating the severity orrapidity of onset of symptoms of the disease. An agent which providesprotection to a lesser degree than do competitive agents may still be ofvalue if the other agents are ineffective for a particular individual,if it can be used in combination with other agents to enhance the levelof protection, or if it is safer than competitive agents.

The effectiveness of a treatment can be determined by comparing theduration, severity, etc. of the disease post-treatment with that in anuntreated control group, preferably matched in terms of the diseasestage.

The effectiveness of a prophylaxis will normally be ascertained bycomparing the incidence of the disease in the treatment group with theincidence of the disease in a control group, where the treatment andcontrol groups were considered to be of equal risk, or where acorrection has been made for expected differences in risk.

In general, prophylaxis will be rendered to those considered to be athigher risk for the disease by virtue of family history, prior personalmedical history, or elevated exposure to the causative agent.

Pharmaceutical Administration

At least one protective agent of the present invention may beadministered by any means that achieve the intended purpose, using apharmaceutical composition as previously described.

Administration may be oral or parenteral, and, if parenteral, eitherlocally or systemically. For example, administration of such acomposition may be by various parenteral routes such as subcutaneous,intravenous, intradermal, intramuscular, intraperitoneal, intranasal,transdermal, or buccal routes. Parenteral administration can be by bolusinjection or by gradual perfusion over time. A preferred mode of using apharmaceutical composition of the present invention is by subcutaneous,intramuscular or intravenous application. See, e.g., Berker, supra,Goodman, supra, Avery, supra and Katzung, supra, which are entirelyincorporated herein by reference, including all references citedtherein.

A typical regimen for preventing, suppressing, or treating a disease orcondition which can be alleviated by an immune response by activespecific immunotherapy, comprises administration of an effective amountof a pharmaceutical composition as described above, administered as asingle treatment, or repeated as enhancing or booster dosages, over aperiod up to and including between one week and about 24 months.

It is understood that the effective dosage will be dependent upon theage, sex, health, and weight of the recipient, kind of concurrenttreatment, if any, frequency of treatment, and the nature of the effectdesired. The ranges of effective doses provided below are not intendedto limit the invention and represent preferred dose ranges. However, themost preferred dosage will be tailored to the individual subject, as isunderstood and determinable by one of skill in the art, without undueexperimentation. This will typically involve adjustment of a standarddose, e.g., reduction of the dose if the patient has a low body weight.See, e.g., Berkow et al, eds., The Merck Manual, 15th edition, Merck andCo., Rahway, N.J., 1987; Goodman et al., eds., Goodman and Gilman's ThePharmacological Basis of Therapeutics, 8th edition, Pergamon Press,Inc., Elmsford, N.Y., (1990); Avery's Drug Treatment: Principles andPractice of Clinical Pharmacology and Therapeutics, 3rd edition, ADISPress, LTD., Williams and Wilkins, Baltimore, Md. (1987), Ebadi,Pharmacology, Little, Brown and Co., Boston, (1985); Chabner et al.,supra; De Vita et al., supra; Salmon, supra; Schroeder et al., supra;Sartorelli et al., supra; and Katsung, supra, which references andreferences cited therein, are entirely incorporated herein by reference.

Prior to use in humans, a drug will first be evaluated for safety andefficacy in laboratory animals. In human clinical studies, one wouldbegin with a dose expected to be safe in humans, based on thepreclinical data for the drug in question, and on customary doses foranalogous drugs (if any). If this dose is effective, the dosage may bedecreased, to determine the minimum effective dose, if desired. If thisdose is ineffective, it will be cautiously increased, with the patientsmonitored for signs of side effects. See, e.g., Berkow, et al., eds.,The Merck Manual, 15th edition, Merck and Co., Rahway, N.J., 1987;Goodman, et al., Goodman and Gilman's The Pharmacological Basis ofTherapeutics, 8th edition, Pergamon Press, Inc., Elmsford, N.Y., (1990);Avery's Drug Treatment: Principles and Practice of Clinical Pharmacologyand Therapeutics, 3rd edition ADIS Press, LTD., Williams and Wilkins,Baltimore, Md. (1987), Ebadi, Pharmacology, Little, Brown and Co.,Boston, (1985), which references and references cited therein, areentirely incorporated herein by reference.

The total dose required for each treatment may be administered inmultiple doses (which may be the same or different) or in a single dose,according to an immunization schedule, which may be predetermined or adhoc. The schedule is selected so as to be immunologically effective,i.e., so as to be sufficient to elicit an effective immune response tothe antigen and thereby, possibly in conjunction with other agents, toprovide protection. The doses adequate to accomplish this are defined as“therapeutically effective doses.” (Note that a schedule may beimmunologically effective even though an individual dose, ifadministered by itself, would not be effective, and the meaning of“therapeutically effective dose” is best interpreted in the context ofthe immunization schedule.) Amounts effective for this use will dependon, e.g., the peptide composition, the manner of administration, thestage and severity of the disease being treated, the weight and generalstate of health of the patient, and the judgment of the prescribingphysician.

Typically, the daily dose of an active ingredient of a pharmaceutical,for a 70 kg adult human, is in the range of 10 nanograms to 10 grams.For immunogens, a more typical daily dose for such a patient is in therange of 10 nanograms to 10 milligrams, more likely 1 microgram to 10milligrams. However, the invention is not limited to these dosageranges.

It must be kept in mind that the compositions of the present inventionmay generally be employed in serious disease states, that is,life-threatening or potentially life threatening situations. In suchcases, in view of the minimization of extraneous substances and therelative nontoxic nature of the peptides, it is possible and may be feltdesirable by the treating physician to administer substantial excessesof these peptide compositions.

The doses may be given at any intervals which are effective. If theinterval is too short, immunoparalysis or other adverse effects canoccur. If the interval is too long, immunity may suffer. The optimuminterval may be longer if the individual doses are larger. Typicalintervals are 1 week, 2 weeks, 4 weeks (or one month), 6 weeks, 8 weeks(or two months) and one year. The appropriateness of administeringadditional doses, and of increasing or decreasing the interval, may bereevaluated on a continuing basis, in view of the patient'simmunocompetence (e.g., the level of antibodies to relevant antigens).

A variety of methods are available for preparing liposomes, as describedin, e.g., Szoka et al., Ann. Rev. Biophys. Bioeng. 9:467 (1980), U.S.Pat. Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369, incorporatedherein by reference.

The appropriate dosage form will depend on the disease, the immunogen,and the mode of administration; possibilities include tablets, capsules,lozenges, dental pastes, suppositories, inhalants, solutions, ointmentsand parenteral depots. See, e.g., Berker, supra, Goodman, supra, Avery,supra and Ebadi, supra, which are entirely incorporated herein byreference, including all references cited therein.

The antigen may be delivered in a manner which enhance, e.g., deliveringthe antigenic material into the intracellular compartment such that the“endogenous pathway” of antigen presentation occurs. For example, theantigen may be entrapped by a liposome (which fuses with the cell), orincorporated into the coat protein of a viral vector (which infects thecell).

Another approach, applicable when the antigen is a peptide, is to injectnaked DNA encoding the antigen into the host, intramuscularly. The DNAis internalized and expressed.

It is also possible to prime autologous PBLs with the compositions ofthe present invention, confirm that the PBLs have manifested the desiredresponse, and then administer the PBLs, or a subset thereof, to thesubject.

EXAMPLES

General: Melting points were not corrected. All air and moisturesensitive reactions were performed under nitrogen atmosphere. AnhydrousTHF, DMF and dichloromethane were purchased from Aldrich and other drysolvents were prepared in the usual way. ACS grade solvents werepurchased from Fisher and used for chromatography without distillation.TLC plates (silica gel 60 F₂₅₄, thickness 0.25 mm, Merck) and flashsilica gel 60 (35-75 mm) for column chromatography were purchased fromRose Scientific, Canada. ¹H and ³¹P spectra were recorded either on aBrucker AM 300 MHz or Varian Unity 500 MHz or Brucker DRX 600 MHzspectrometers with TMS as internal standard for proton chemical shifts.Optical rotations were measured on a Perkin-Elmer 241 Polarimeter atroom temperature (20-22° C.). Elemental analysis data were obtained fromthe Micro-analytical laboratory in the University of Alberta.Electron-spray mass spectrometric analyses were performed either on aMS50B or MSD1 SPC mass spectrometers.

Example 1 Preparation of Compound 8

Compound 6 (312 mg, 0.65 mmol), 7 (200 mg, 0.44 mmol), DCC (136 mg, 0.66mmol) and DMAP (27 mg, 0.22 mmol) were dissolved in dry dichloromethane(5 ml). The mixture was stirred at room temperature for 4 h. The solidwas filtered off and washed with ethyl acetate (5 ml). The filtrate wasconcentrated and the residue was purified by flash chromatography(hexane:ethyl acetate, 8:1) to give 8 (398 mg, 98%). TLC: R_(f)=0.69(hexane:ethyl acetate, 3:1). [a]_(D) ²²=+32.0 (c 0.5, chloroform). ¹HNMR (300 MHz, CDC1₃): d 0.90 (t, J=6.5 Hz, 6H, 2 CH₃), 1.25 (m, 38 H, 19CH₂), 1.52 (m, 4H, 2 CH₂), 2.16 (t, J=7.5 Hz, 2H, CH₂), 2.50 (dd,J=16.0, 6.0 Hz, 1H, CHH), 2.63 (dd, J=16.0, 6.0 Hz, 1H, CHH), 3.71 (dd,J=9.5, 9.5 Hz, 1H, H-4), 3.78 (dd, J=10.0, 10.0 Hz, 1H, H-6a), 3.94 (m,1H, H-5), 3.98-4.08 (m, 2H, H-2, CHHCH═CH₂), 4.21 (m, 1H, CHHCH═CH₂),4.29 (dd, J=10.0, 5.0 Hz, 1H, H-6b), 4.69, 4.76 (2 d, J=12.0 Hz, each1H, Troc-CH₂), 4.94 (d, J=3.6 Hz, 1H, H-1), 5.16 (m, 1H, lipid-3-H),5.30 (m, 2H, CH═CH₂), 5.39 (dd, J=9.5, 9.5 Hz, 1H, H-3), 5.42 (d, J=10.0Hz, 1H, NH), 5.53 (s, 1H, CHPh), 5.90 (m, 1H, CH═CH₂), 7.30-7.35 (m,15H, Ar—H). Anal. calcd for C₄₇H₇₄Cl₃NO₁₀ (919.46):C, 61.40; H, 8.11; N,1.52. Found: C, 61.40; H, 8.19; N, 1:58.

Example 2 Preparation of Compound 9

To a solution of 8 (1.45 g, 1.60 mmol) in dry THF (20 ml) was addedmolecular sieves (4 A, 3.0 g). The mixture was stirred at roomtemperature under nitrogen for 20 min. Sodium cyanoborohydride (1.0 g,15.96 mmol) was added and the mixture was cooled to 0° C. HCl (g)/Et₂Osolution was added drop wise slowly till no gas was evolved. The mixturewas then poured into saturated sodium bicarbonate solution (50 ml) andextracted with dichloromethane (100 ml×3). Combined organic layers werewashed with saturated sodium chloride solution (20 ml) and dried withsodium sulfate, and concentrated. The residue was purified by flashsilica gel chromatography (initially with hexane:ethyl acetate, 5:1 andthen 4:1) to give 9 (1.23 g, 85%). TLC: R_(f)=0.20 (hexane:ethylacetate, 4: 1). [a]_(D) ²⁰=+47.5 (c 1.0, CHC1₃). ¹H NMR (300 MHz,CDC1₃): d 0.88 (t, J=6.5 Hz, 6H, 2 CH₃), 1.25 (br s, 38H, 19 CH₂), 1.50(m, 4H, 2 CH₂), 2.28 (t, J=7.5 Hz, 2H, CH₂), 2.48 (dd, J=14.0, 4.0 Hz,1H), 2.58 (dd, J=14.0, 7.5 Hz, 1H), 3.27 (d, J=3.5 Hz, 1H, OH),3.70-3.86 (m, 4H), 3.92-4.03 (m, 2H), 4.58 (d, J=12.0 Hz, 1H), 4.64 (d,J=12.0 Hz, 1H), 4.66 (d, J=12.0 Hz, 1H), 4.76 (d, J=12.0 Hz, 1H), 4.92(d, J=3.5 Hz, 1H, H-1), 5.13 (m, 2H), 5.19-5.31 (m, 2H, CH₂═CH), 5.40(d, J=9.5 Hz, 1H, NH), 5.88 (m, 1H, CH₂═CH), 7.30 (m, 5H, Ar—H). ES-MScalcd for C₄₇H₇₆Cl₃NO₃: 919.5. Found: 920.8 (M+H).

Example 3 Preparation of Compound 10

To compound 9 (1.20 g, 1.30 mmol) in dry dichloromethane (20 ml) wereadded 1H-tetrazole (273 mg, 3.90 mmol) and dibenzyldiisopropylphosphoramidite (900 mg, 0.875 ml, 2.61 mmol). The mixturewas stirred at room temperature for 30 min and then cooled to 0° C.m-Chloroperbenzoic acid (m-CPBA, 1.63 g, 55%, 5.22 mmol) was added andthe mixture was stirred for 30 min at 0° C. The mixture was then pouredinto 10% sodium hydrogen sulfite (40 ml) and extracted withdichloromethane (40 ml×3). The organic layer was washed with saturatedsodium bicarbonate solution (20 ml), dried with sodium sulfate andconcentrated. The residue was purified by repeated flash chromatography(initially hexane:ethyl acetate, 4: 1 and then 3: 1). TLC: R_(f)=0.31(hexane:ethyl acetate, 3:1) to give 10 (1.33 g, 86%). [a]_(D) ²⁰=+35.0(c 1.0, CHC1₃). ¹H NMR (300 MHz, CDC1₃): d 0.88 (t, J=6.5 Hz, 6H, 2CH₃), 1.24 (br s, 38H, 19 CH₂), 1.50 (m, 4H, 2 CH₂), 2.17 (t, J=7.0 Hz,2H, CH₂), 2.41 (dd, J=16.5, 5.5 Hz, 1H), 2.51 (dd, J=16.5, 7.5 Hz, 1H),3.66 (dd, J=11.0, 4.5 Hz, 1H), 3.74 (dd, J=11.0, 2.0 Hz, 1H), 3.91 (m,1H), 4.00 (m, 2H), 4.20 (m, 1H), 4.44 (d, J=12.0 Hz, 1H), 4.53 (m, 1H,H-4), 4.54 (d, J=12.0, 1H), 4.63 (d, J=12.0, 1H), 4.88-4.95 (m, 5H),5.11 (m, 1H), 5.20-5.32 (m, 2H, CH₂═CH), 5.35 (dd, J=10.5, 9.0 Hz, 1H,H-3), 5.41 (d, J=9.5 Hz, 1H, NH), 5.88 (m, 1H, CH₂═CH), 7.30 (m, 15 H,Ar—H). ES-MS calcd for C₆₁H₈₉C₁₃NO₁₃P: 1179.6, Found: 1181.0 (M+H).

Example 4 Preparation of Compound 11

[Bis(methyldiphenylphosphine)](1,5-cyclooctadiene) iridium (I)hexafluorophosphate (14 mg, 0.0165 mmol) was suspended in dry THF (5 ml)and hydrogen gas was bubbled in for 5 min to give a yellowish solution,which was added to the solution of 10 (1.30 g, 1.10 mmol) in dry THF (10ml). The mixture was stirred at room temperature for 2 hours. Water (0.5ml) and N-bromosuccinimide (NBS, 294 mg, 1.62 mmol) were then added andthe reaction was stirred for 1 hour longer. Remainder obtained fromsolvent removal was dissolved in ethyl acetate (200 ml) and washed withsaturated sodium bicarbonate solution (20 ml.×2). Combined organiclayers were dried with sodium sulfate and concentrated. The residue waspurified by flash chromatography (hexane:ethyl acetate, 2: 1) to give 11(950 mg, 76%). TLC: R_(f)=0.31 (ethyl acetate:hexane, 1: 2). [a]_(D)²⁰=+17.5 (c 1.0, CHC1₃). ¹H NMR (300 MHz, CDC1₃): δ 0.88 (t, J=6.5 Hz,6H, 2 CH₃), 1.24 (br s, 38H, 19 CH₂), 1.59 (m, 0.4H, 2 CH₂), 2.18 (t,J=7.0 Hz. 2 J. CH₂), 2.39 (m, 2H, CH₂), 3.59 (dd, J=11.0, 6.0 Hz, 1H),3.71 (dd, J=11.0, 1.5 Hz, 1H), 3.94 (m, 1H), 4.16 (m, 1H), 4.40 (m, 3H),4.49 (d, J=12.0 Hz, 1H), 4.65 (d, J=12.0 Hz, 1H), 4.72 (d, J=12.0 Hz,1H), 4.90 (m, 4H), 5.09 (m, 1H), 5.39 (t, J=3.5 Hz, 1H, H-1), 5.37 (dd,J=10.0, 9.5 Hz, 1H, H-3), 5.70 (d, J=9.5 Hz, 1H, NH), 7.30 (m, 15H,Ar—H). ES-MS calcd for C₅₈H₈₅C1₃NO₁₃P:

1139.5. Found: 1141.0 (M+H).

Example 5 Preparation of Compound 12

To a solution of 11 (920 mg, 0.81 mmol) in dry dichloromethane (10 ml),trichloroacetonitrile (2 ml) and DBU (4 drops) were added. The mixturewas stirred at room temperature for 2 h and concentrated in vacuo (notto dryness). The residue was purified by flash chromatography(hexane:ethyl acetate, 4:1, 3.5:1 and 3:1, with 0.5% of triethyl amine)to give 12 (700 mg, 68%). TLC: R_(f)=0.36 (hexane:ethyl acetate, 3:1).[a]_(D) ²⁰=+12.5 (c 0.4, CHC1₃). ¹H NMR (300 MHz, CDC1₃): δ 0.88 (t,J=6.5 Hz, 6H, 2 CH₃), 1.24 (br s, 38H, 19 CH₂), 1.50 (m, 4H, 2 CH₂),2.19 (t, J=7.0 Hz, 2H, CH₂), 2.46 (m, 2H, CH₂), 3.71 (m, 2H), 4.04 (m,1H). 4.15 (ddd, J=1.0, 8.5, 3.5 Hz, 1H, H-2), 4.43 (d, J=12.0 Hz, 1H),4.52 (d, J=12.0 Hz, 1H), 4.61 (d, J=12.0 Hz, 1H), 4.71 (ddd, J=9.5, 9.5,9.5 Hz, 1H, H-4), 4.77 (d, J=12.0 Hz, 1H). 4.94 (m, 4H), 5.12 (m, 1H),4.39 (dd, J=10.0, 9.5 Hz, 1H, H-3), 5.65 (d, J=8.5 Hz, 1H, NH), 6.47 (d,J=3.5 Hz, 1H, H-1), 7.32 (m, 15H, Ar—H), 8.72 (s, 1H, NH). ES-MS calcdfor C₆₀H₈₅Cl₆N₂O₁₃P: 1282.4. Found: 1284.0 (M+H).

Example 6 Preparation of Compound 14

Compound 13 (672 mg, 2.97 mmol) was dissolved in dry acetonitrile (10ml) and 2,2-dimethoxypropane (560 mg, 0.66 ml, 5.35 mmol) andp-toluenesulfonic acid (56 mg, 0.279 mmol) were added. The mixture wasstirred at room temperature for 1 h and then triethylamine (0.5 ml) wasadded to quench the reaction. The mixture was concentrated in vacuo andthe residue purified by flash chromatography (hexane/ethyl acetate,2:1). to give 14 (614 mg, 82%). R_(f)=0.67 (hexane/ethyl acetate, 1:2).¹H NMR (300 MHz, CDC1₃): δ=1.41 (s, 3H, CH₃), 1.42 (s, 3H, CH₃), 2.40(br s, 1H, OH), 3.59 (s, 2H, CH₂), 3.69 (s, 2H, CH₂), 3.74 (s, 4H, 2CH₂), 4.55 (s, 2H, CH₂Ph), 7.30 (m, 5H, Ar—H).

Example 7 Preparation of Compound 15

Compound 14 (572 mg, 2.26 mmol) was dissolved in dry pyridine (3 ml) andcooled to 0° C. P-Toluenesulfonyl chloride (5.7 mg, 2.71 mmol) was addedand the mixture was stirred for 3 h. More toluenesulfonyl chloride (430mg, 2.26 mmol) was added and the reaction mixture was stirred at roomtemperature overnight. Methanol (1 ml) was then added to quench thereaction and the solvent was removed in vacuo by co-distillation withtoluene. The residue was dissolved in dichloromethane (100 ml) andwashed with sat. NaHCO₃ (aq.) (30 ml). The aqueous layer was extractedwith dichloromethane (30 ml) and the combined organic layer was driedwith sodium sulfate and concentrated. The residue was purified by flashchromatography (hexane/ethyl acetate, 5:1) to give 15 (930 mg, 98%).R_(f)=0.65 (hexane/ethyl acetate, 2:1). ¹H NMR (300 MHz, CDC1₃): δ=1.30(s, 3H, CH₃), 1.40 (s, 3H, CH₃), 2.42 (s, 3H, CH³), 3.35 (s, 2H, CH₂),3.63 (d, J=12.0 Hz, 2 H), 3.72 (d, J=12.0 Hz, 2H), 4.20 (s, 4H, 2 CH₂),4.50 (s, 2 H, CH₂Ph), 7.30 (m, 7H, Ar—H), 7.78 (m, 2H, Ar—H). ES-MScalcd for C₂₂H₂₈O₆S: 420.2; found: 443.2 (M+Na).

Example 8 Preparation of Compound 16

Compound 15 (907 mg, 2.16 mmol) was dissolved in toluene (30 ml) andsat. NaHCO3 (aq.) (30 ml), sodium azide (561 mg, 8.63 mmol), and phasetransfer catalyst ALIQUAT (433 mg, 0.49 ml, 1.08 mmol) were added. Themixture was refluxed for 16 h and more sodium azide (1.40 g, 21.60 mmol)was added. The reaction was continued for 24 h and then cooled to roomtemperature. The organic layer was separated and the aqueous layer wasextracted with ethyl acetate (30 ml×3). The combined organic layer waswashed with water (30 ml), dried with sodium sulfate, and concentratedin vacuo. The residue was purified by flash chromatography (hexane/ethylacetate, 8:1) to give 16 (440 mg, 70%) and the starting material 15 (163mg, 18%). R_(f)=0.34 (hexane/ethyl acetate, 6: 1). ¹H NMR (500 MHz,CDC1₃): δ=1.42 (s, 6H, 2 CH₃), 3.40 (s, 2H, CH₂), 3.52 (s, 2H, CH₂),3.64 (d, J=12.0 Hz, 2H), 3.73 (d, J=12.0 Hz, 2H), 4.50 (s, 2H, CH₂Ph),7.30 (m, 5H, Ar—H). ES-MS calcd for C₁₆H₂₁N₃O₃:291.2; found: 314.1(M+Na). ES-MS calcd for C₁₅H₂₁N₃O₃: 291.2; found: 314.1 (M+Na).

Example 9 Preparation of Compound 17

Compound 16 (40 mg, 0.137 mmol) was dissolved in acetic acid (10 ml) andzinc powder (1.0 g) was added. The mixture was stirred at roomtemperature for 1 h and the solid was filtered out and washed withacetic acid (10 ml). The filtrate was concentrated in vacuo. The residuewas dissolved in dioxane-sat. NaHCO₃ (aq.) (2:1, 6 ml, PH 8-9) and2,2,2-trichloroethoxylchloroformate (123 mg, 0.08 ml, 0.568 mmol) wasadded. The mixture was stirred at room temperature for 6 h. The dioxanewas then removed in vacuo and water (10 ml) was added. The mixture wasextracted with ethyl acetate (10 ml×3) and the organic layer was driedwith sodium sulfate and concentrated in vacuo. The residue was purifiedby flash chromatography (hexane/ethyl acetate, 4:1) to give 17 (28 mg,47%). R_(f)=0.17 (hexane/ethyl-acetate, 6: 1). ¹H NMR (300 MHz, CDC1₃):δ=1.39 (s, 3H, CH₃), 1.41 (s, 3 H, CH₃), 3.32 (d, J=6.0 Hz, 2H, CH₂),3.54 (s, 2H, CH₂), 3.67 (d, J=12.0 Hz, 2H), 3.75 (d, J=12.0 Hz, 2H),4.57 (s, 2H), 4.72 (s, 2H), 5.50 (t, J=6.0 Hz, 1H, NH), 7.35 (m, 5H, H).ES-MS calcd for C₁₈H₂₄Cl₃NO₅: 439.1; found: 462.1 (M+Na), 464.1 (M+Na,³⁷C1).

Example 10 Preparation of Compound 18

Compound 17 (18.3 mg, 0.0417 mmol) was dissolved in acetic acid-water(4:1, 10 ml) and treated at 60° C. for 45 min. The solvent was removedin vacuo and the residue was purified by flash chromatography(hexane/ethyl acetate, 1:1) to give 18 (15 mg, 90%). ¹H NMR (300 MHz,CDC1₃): δ=3.03 (t, J=6.5 Hz, 2H, 2 OH), 3.43 (s, 2H, CH₂), 3.44 (d,J=6.5 Hz, 2H, CH₂NH), 3.51 (d, J=6.5 Hz, 4H, 2 CH₂OH), 4.55 (s, 2H),4.73 (s, 2H), 5.35 (t, J=6.5 Hz, 1H, NH), 7.35 (m, 5H, Ar—H). ES-MScalcd for C₁₅H₂₀C1₃NO₅: 399.0; found: 422.0 (M+Na), 424.0 (M+Na, ³⁷Cl).

Example 11 Preparation of Compound 19

To a solution of 12 (620 mg, 0.484 mmol) and 18 (750 mg, 1.936 mmol) indry dichloromethane (15 ml) was added molecular sieves (4A, 2.0 g) andthe mixture was stirred under nitrogen for 10 min at room temperature.Trimethysilyl trifluoromethanesulfonate (TMSOTf) solution (0.01 M indichloromethane) (3.0 ml) was added drop wise within 5 min. The mixturewas stirred at room temperature for 1 h and saturated sodium bicarbonatesolution (10 ml) was added to quench the reaction. Usual aqueous work-upand flash chromatography (hexane/acetone, 2.8:1 and 2:1) afforded 19(590 mg, 81) as a diastereomeric mixture in a ratio of about 1:1.R_(f)=0.27 (hexane/acetone, 2.5:1). [a]_(D) ²⁰=−7.6 (c 0.8, chloroform).¹H NMR (300 MHz, CDC1₃): d=0.88 (t, J=6.5 Hz, 6 H, 2 CH₃), 1.25 (br s,36H, 18 CH₂), 1.45 (m, 2H, CH₂), 1.58 (m, 2H, CH₂), 1.68 (m, 2H, CH₂),2.23 (t, J=7.5 Hz, 2H, CH₂). 2.41 (m, 2H, CH₂), 3.10 (m, 0.5H),3.30-3.62 (m, 7.5 Hz). 3.68-3.83 (m, 2H), 4.40-4.56 (m, 7H), 4.65-4.80(m, 5H), 4.90 (m, 5H), 5.19 (m, 2H), 5.53 (d, J=9.0 Hz, 0.5H, NH), 5.72(m, 1.5H, NH), 7.30 (m, 20H, Ar—H). ES-MS calcd for C₇₃H₁₀₃C₁₆N₂O₁₇P:1520.5; found: 1543.5 (M+Na, 42), 1544.4 (M+Na, ¹³C-isotope 34), 1545.5(M+Na, ³⁷C1-isotope, 100).

Example 12 Preparation of Compound 20

Compound 19 (450 mg, 0.30 mmol) was dissolved in acetic acid (50 ml) andzinc power (4.0 mg) was added. The mixture was stirred at roomtemperature for 1 h and the solid was filtered out. The solid wasfurther washed with acetic acid (50 ml) and the filtrate wasconcentrated in vacuo. The residue was dissolved in dichloromethane (150ml) and the solution was washed with saturated aqueous sodiumbicarbonate solution (20 ml). The aqueous layer was back washed withdichloromethane (20 ml×2). The combined organic layer was dried withsodium sulphate and concentrated in vacuo to give the di-amineintermediate (346 mg). A mixture of the di-amine (346 mg) and lipid acid7 (545 mg, 1.20 mmol) and DCC (371 mg, 1.80 mmol) in dry dichloromethane(10 ml) was stirred at room temperature for 20 h. Water (0.05 ml) wasadded and the reaction mixture was stirred for 10 min. The solid wasfiltered out through a sintered glass funnel bedded with sodiumsulphate. The filtrate was concentrated and the residue purified byflash chromatography (hexane/acetone, 5: 1 and 4.5: 1) to give 20 (390mg, 64%). R_(f)=0.20 (hexane/acetone, 4:1). [a]_(D) ²⁰=−9.4 (c 0.5,chloroform). ¹H NMR (500 MHz, CDC1₃): d=0.88 (t, J=7.0 Hz, 18H, 6 CH₃),1.25-1.50 (m, 112H, 56 CH₂), 1.58 (m, 11H), 1.71-1.81 (m, 3H), 1.97 (m,1H, OH), 2.21 (t, J=7.5 Hz, 2H, CH₂), 2.24-2.63 (m, 10H, 5 CH₂), 3.06(dd, J=14.0, 5.0 Hz, 0.5H), 3.17 (dd, J=14.0, 6.0 Hz, 0.5 Hz), 3.30-3.40(m, 3H), 3.43-3.53 (m, 2H), 3.57-3.63 (m, 2.5H), 3.67-3.80 (m, 2H), 3.87(m, 1H), 3.97 (m, 0.5H), 4.35 (d, J=8.0 Hz, 0.5H, H-1), 4.38-4.53 (m,5H), 4.65 (d, J=8.0 Hz, 0.5H, H-1), 4.90 (m, 4H), 5.07-5.24 (m, 4H),6.04 (d, J=8.5 Hz, 0.5H, NH), 6.38 (d, J=7.5 Hz, 0.5H, NH), 6.65 (dd,J=6.5, 6.5 Hz, 0.5H, NH), 6.79 (dd, J=7.0, 6.0 Hz, 0.5H, NH), 7.30 (m,20H, Ar—H). ES-MS calcd. for C₁₂₃H₂₀₅N₂O₁₉P: 2045.5; found: 2068.5(M+Na, 63). 2069.5 (M+Na, ¹³-isotope, 100).

Example 13 Preparation of Compound 21

To a solution of compound 20 (220 mg, 0.108 mmol) in dry dichloromethane(5 ml) were added dibenzyl diisopropyl phosphoramidite (74.3 mg, 74.3μl, 0.215 mmol) and 1H-tetrazole (22.7 mg, 0.324 mmol). The mixture wasstirred at room temperature for 30 min and then cooled to 0° C.m-Chloroperbenzoic acid (m-CPBA, 55%, 118 mg, 0.379 mmol) was added andthe mixture was stirred at 0° C. for 30 min. The mixture was dilutedwith dichloromethane (100 ml) and washed with aqueous sodium bisulphitesolution (10%, 20 ml) and the aqueous layer was extracted withdichloromethane (20 ml). The combined organic layer was then washed withsaturated sodium bicarbonate solution (20 ml) and aqueous layer was backwashed with dichloromethane (20 ml). The combined organic layer was thendried with sodium sulphate and concentrated in vacuo. The residue waspurified by repeated flash chromatography (hexane/acetone, 5: 1 and then4.5: 1; dichloromethane/methanol, 100:1 and then 100:1.5; hexane/ethylacetate, 2:1 and then 1.5:1) to give 21 (200 mg, 80%) as adiastereomeric mixture in a ratio of about 1:1. R_(f) (upper spot)=0.29and R_(f) (lower spot)=0.25 (hexane/acetone, 3:1). [a]_(D) ²⁰ (˜1:1mixture)=−7.6 (c 0.5, chloroform). ¹H NMR (300 MHz, CDCl₃): d=0.87 (t,J=6.5 Hz, 18H, 6 CH₃), 1.30 (m, 108H, 54 CH₂), 1.48-1.70 (m, 18H),2.10-2.53 (m, 11H), 2.90-3.35 (m, 5H), 3.55 (m, 2H), 3.75-3.90 (m, 4H),4.00 (m, 1H), 4.36-4.52 (m, 6H), 4.85-5.01 (m, 8H), 5.08-5.22 (m, 4H),6.30 (m, 1H, NH), 6.88 (d, J=8.5 Hz, 0.5H, NH), 7.00 (d, J=8.0 Hz, 0.5H,NH), 7.30 (m, 30 H, Ar—H). ES-MS calcd. for C₁₃₇H₂₁₈N₂O₂₂P: 2305.5;found: 2328.5 (M+Na, 78), 2329.5 (M+Na, ¹³C-isotope, 100).

Example 14 Preparation of Compound 1

Compound 20 (96 mg, 0.047 mmol) was dissolved in THF-HOAc (10:1, 77 ml)and palladium on charcoal (100 mg) was added. The mixture was stirredunder hydrogen atmosphere for 24. The solid was then filtered out andwashed with chloroform/methanol (1:1, 30 ml). The filtrate wasconcentrated in vacuo and the residue purified by flash chromatography(chlotoform/methanol/water, 9:1:0 and then 4:1:0.1) to give 1 which wasfreeze dried from tert-butanol to afford the product as white powder (80mg, 100%). R_(f)=0.16 (chloroform/methanol/water/acetic acid, 6:1:0.1:0.1). [a]_(D) ²⁰=−6.5 (c 0.2, chloroform). ¹H NMR (600 MHz,CDC1₃—CD₃OD, 1:1): d=0.89 (t, J −6.5 Hz, 18H, 6 CH₃), 1.26 (m, 114H,57H), 1.60 (m, 12H, 6 CH₂), 2.30 (m, 6H, 3 CH₂). 2.37 (dd, J=15.0, 6.0Hz, 1H), 2.45 (dd, J=15.0, 7.0 Hz, 1H), 2.50 (dd, J=15.0, 5.0 Hz, 1H),2.54 (dd, J=15.0, 8.0 Hz, 1H), 2.57H), (dd, J=15.0, 5.0 Hz, 1H), 2.67(dd, J=15.0, 7.0 Hz 1H), 3.04 (dd, J=14.0, 6.0 Hz, 1H), 3.18 (dd,J=14.0, 6.0 Hz, 1H), 3.25 (d, J=10.0 Hz, 1H), 3.29 (d, J=10.0 Hz, 1H),3.36 (m, 3H), 3.37 (d, J=10.0 Hz, 1H), 3.64 (d, J=10.0 Hz, 1H), 3.77 (brd, J=12.0 Hz, 1H), 3.89 (dd, J −10.0, 9.0 Hz, 1H), 3.96 (br d, J=12.0Hz, 1H), 4.25 (m, 1H, H-4), 4.43 ((d, J=8.5 Hz, 1H, H-1), 5.07 (dd,H=10.0, 10.0, Hz, 1H, H-3), 5.17 (m, 2H), 5.23 (m, 1H). ES-MS calcd. forC₉₅H₁₈₁N₂O₁₉P: 1685.3; found: 1686.3 (M+H), 1708.3 (M+Na), 1730.3(M+2Na−H).

Example 15 Preparation of Compound 2

In a similar was as described for 1, compound 21 (104 mg, 0.045 mmol)was treated with palladium on charcoal (100 mg) in THF-HOAc (10:1, 77ml) under hydrogen atmosphere for 20 h to give 2 (77 mg, 96%) afterflash chromatography purification (chloroform/methanol/water, 9:1:0 andthen 6:4:0.5). R_(f)=0.50 (chloroform/methanol/water, 6:4:0.5). [a]_(D)²⁰=−3.0 (c 0.2, chloroform). ¹H NMR (600 MHz, CDC1₃-CD₃OD, 1:1): d=0.89(t, J=6.5 Hz, 18H, 6 CH3), 1.25 (m, 114H, 57 CH2), 1.60 (m, 12H, 6 CH2),2.30H, 6H, 3 CH2), 2.37-2.70 (m, 6H, 3 CH2), 3.03 (d, J=14.0 Hz, 0.5H),3.13 (d, J=14.0 Hz, 0.5H), 3.24 (d, J=14.0 Hz, 0.5H), 3.27 (d, J=14.0Hz, 0.5H), 3.29-3.36 (m, 2H), 3.45 (br s, 1H), 3.55-3.95 (m, 6H),4.06-4.32 (m, 2H), 5.14-5.27 (m, 4H). ES-MS calcd. for C₉₅H₁₈₁N₂O₁₉P:1685.3; found: 1686.3 (M+H), 1708.3 (M+Na), 1730.3 (M+2Na —H).

Example 16 Preparation of Compound 22

To Dipentaerythritol (2.0 g, 7.87 mmol) in dry DMF (10 mL) were addedbenzaldehyde dimethyl acetal (4.79 g, 4.7 ml, 31.46 mmol) andtoluenesulfonic acid (150 mg, 0.78 mmol) and the mixture was stirred at50° C. for 1 h. TLC (methanol: dichloromethane, 8:92) showed product andupper impurity, thought to be other —OH sites also substituted. Tohydrolyze upper impurity, triethylamine (5 drops) was added toneutralize, DMF was evaporated under high vacuum, and methanol wasadded. The second TLC showed upper spot disappeared. The mixture wasconcentrated to clear syrup and purified by silica gel chromatography(methanol: dichloromethane, 5:95) to give 22 (1.91 g, 57%) as a mixtureof three stereoisomerisms. TLC: R_(f)=0.38 (upper spot) and R_(f)=0.34(lower spot) (7% methanol in dichloromethane). ¹H NMR (500 MHz, CDC1₃):δ=3.21, 3.31, 3.39 (3 s, 4H); 3.28, 3.32, 3.46, 3.51 (4 br s, 2H, 2 OH);3.68, 3.69, 3.75 (3 d, J=12.0 Hz, 4H); 3.81, 3.92, 3.98 (3 s, 4H); 4.10(m, 4H); 5.39, 5.41, 5.43 (3 s, 2H, 2 CHPh), 7.38 (m, 6H, Ar—H); 7.50(m, 4H, Ar—H). Through repeated chromatography(methanol/dichloromethane, gradient elution from 1% to 10%), the lowerspot was separated to give a single component. ¹H NMR (300 MHz, CDC1₃)for the lower spot: δ=2.50 (br s, 2H, 2 OH), 3.45 (br s, 4H), 3.73 (d,J=12.0 Hz, 4H), 3.97 (s, 4H), 4.13 (d, J=12.0 Hz, 4H), 5.43 (s, 2H, 2CHPh), 7.37 (m, 6H, Ar—H), 7.49 (m, 4H, Ar—H).

Example 17 Preparation of Compound 23

Sodium hydride (0.426 g, 17.7 mmol) was added to dry DMF (35 ml) andcooled to 0° C. 22 (1.524 g, 3.54 mmol, dissolved in 15 ml DMF) wasadded drop wise and the mixture was stirred at 0° C. for 30 min foralkoxide formation. Drop wise added n−1-bromo-tetradecane (3.16 ml, 10.6mmol, dissolved in 5 ml DMF) to mixture and stirred at room temperaturefor 16 hrs. TLC (hexane:ethyl acetate, 15:1) showed considerable amountof lower impurity thought to be mono-substitution of lipid arm. Another2 equivalents of sodium hydride (0.21 g) and 2 equivalents ofn−1-bromo-tetradecane (2.1 mL) were added and the mixture was stirredfor 5 hrs at room temperature. More DMF (40 ml) was added to the slurrymixture and the reaction was continued for 16 hrs more at 50° C.Excessive NaH was quenched with water (3 ml) and the reaction mixturewas neutralized with HCl (conc.). Evaporated off DMF, withco-evaporation with toluene (2×30 mL). The residue was dissolved insaturated sodium chloride (100 mL), extracted with dichloromethane(3×100 mL), and back-washed with saturated sodium chloride (30 mL).Dried with sodium sulfate and concentrated. The solid was purified bysilica gel chromatography (hexane:ethyl acetate, 15:1) to give 23 (2.09g, 72%). TLC indicated two spots, which were separated. The upper spotcontains two components and the lower spot is a single compound. 23(upper spot):TLC:R_(f)=0.43 (hexane:ethyl acetate, 15:1). ¹H NMR (500MHz, CDC1₃): δ=0.88 (t, J=6.5 Hz, 6H, 2 CH₃); 1.26 (br s, 44H, 22 CH2);1.54 (m, 4H, 2 CH2); 3.23, 3.24, 3.33 (3 s, 4H); 3.34, 3.37, 3.45 (3 t,J=6.5 Hz, 4H); 3.71, 3.72, 3.81 (3 s, 4H); 3.90 (m, 4H); 4.10 (m, 4H);5.41, 5.43 (2 s, 2H, CHPh), 7.35 (m, 6H, Ar—H); 7.49 (m, 4H, Ar—H). 23(lower spot):TLC:R_(f)=0.36 (hexane:ethyl acetate, 15:1). ¹H NMR (500MHz, CDCl3): δ=0.88 (t, J=6.5 Hz, 6H, 2 CH₃), 1.26 (br s, 44H, 22 CH₂),1.58 (m, 4H, 2 CH₂), 3.23 (s, 4H). 3.47 (t, J=6.5 Hz, 4H, 2 OCH₂CH₂),3.72 (s, 4H), 3.87 (d, J=11.5 Hz, 4H), 4.11 (d, J=11.5 Hz, 4H), 5.43 (s,2H, 2 CHPh), 7.36 (m, 6H, Ar—H), 7.49 (m, 4 H Ar—H).

Example 18 Preparation of Compound 24

To a solution of 23 (0.611 g, 0.742 mmol) in dry THF (20 mL) was addedmolecular sieves (4 Å, 2 g). The mixture was stirred at room temperatureunder nitrogen for 15 min. Sodium cyanoborohydride (0.932 g, 14.84 mmol)was added in portions and the mixture was cooled to 0° C. TFA (3.40 ml,29.68 mmol) dissolved in THF (60 mL) was added drop wise slowly over 45min. and allowed to stir at room temperature for 4 hrs. The mixture wasfiltered over celite and evaporated off THF under vacuum. The mixturewas dissolved into saturated sodium bicarbonate solution (75 mL) andextracted with ethyl acetate (3×75 mL). The combined organic layers werewashed with saturated sodium chloride solution (50 mL) and dried withsodium sulfate, and concentrated to yellow syrup. The syrup was purifiedby chromatography (gradient elution with hexane:ethyl acetate, 5: 1 to3:1) to give compound 24 (364 mg, 59%). TLC:R_(f).=0.19 (hexane:ethylacetate, 4:1). ¹H NMR (300 MHz, CDC1₃): δ=0.89 (t, J=6.5 Hz, 6H, 2 CH3),1.27 (br s, 44H, 22 CH2), 1.52 (m, 4H, 2 CH2), 2.97 (br s, 2H, 2 OH),3.36 (t, J=6.5 Hz, 4H, 2 OCH₂CH₂), 3.44 (s, 8H), 3.48 (s, 4H), 3.69 (brs, 4H), 4.49 (s, 4H, 2 CH₂Ph), 7.30 (m, 10H, Ar—H).

Example 19 Preparation of Compound 25

To compound 24 (247 mg, 0.299 mmol) in dry dichloromethane (5 ml) wereadded 1H-tetrazole (0.136 g, 1.194 mmol) and dibenzyldiisopropylphosphoramidite (0.310 g, 0.3 ml, 0.896 mmol). The mixturewas stirred at room temperature for 1 hour and the formation of complexchecked with TLC (hexane:ethyl acetate, 4: 1, showing 24 consumed). Themixture was then cooled to 0° C. and m-Chloroperbenzoic acid was addedslowly resulting in gas formation. After 30 min, the mixture was pouredinto 10% sodium hydrogen sulfite (40 ml) and extracted withdichloromethane (3×40 ml). The organic layer was washed with saturatedsodium bicarbonate solution (20 ml), dried with sodium sulfate andconcentrated to yellow syrup. The syrup was purified by repeatedchromatography (hexane:ethyl acetate, 3:1; hexane:acetone, 6: 1) to give25 (248 mg, 62%). TLC:R_(f)=0.21 (hexane:ethyl acetate, 2:1). ¹H NMR(400 MHz, CDC1₃): δ=0.88 (t, J=6.5 Hz, 6H, 2 CH₃), 1.22 (br s, 22H, 11CH₂), 1.24 (br s, 22H, 11 CH₂), 1.44 (m, 4H, 2 CH₂), 3.26 (t, J=6.5 Hz,4H, 2 OCH₂CH₂), 3.32 (s, 4H), 3.34 (s, 4H), 3.40 (s, 4H), 4.07 (d, J=3.5Hz, 4H), 4.38 (s, 4H), 4.99 (d, J=8.0 Hz, 8H, 4 CH₂Ph), 7.30 (m, 30H,Ar—H).

Example 20 Preparation of Compound 3

To a solution of 25 (150 mg, 0.111 mmol) in THF-HOAc (10:1, 90 mL) wasadded palladium on carbon (5%, 105 mg). The mixture was stirred at roomtemperature under hydrogen atmosphere for 16 hrs. TLC(chloroform:methanol: water: acetic acid, 4:1:0.1: 0.1) indicatedpartial hydrogenation. Additional THF-HOAc (60 mL) and Pd/C (100 mg)were added to the mixture and allowed to stir at room temperature underhydrogen atmosphere over second night. TLC (chloroform:methanol:ammoniumhydroxide:water, 1: 1:8%:8%) indicated mostly product. The solid wasfiltered off and the filtrate concentrated under high vacuum. Theresidue was purified by chromatography (chloroform:methanol: ammoniumhydroxide: water, 4:6:6% 6% to 1:1:8%:8%) to give 3 as ammonium salt.The product was re-dissolved in CHCl₂—MeOH—H₂O (1:1:8%) and passedthrough a small ion-exchange column (IR-120, Na⁺ form) to give 3 (44 mg,47%) as sodium salt. TLC:R_(f)=0.41 (chloroform:methanol: ammoniumhydroxide: water, 1:1:8%:8%). ES-MS calculated for C₃₈H₈₀O₁₃P₂: 806.5.Found: 805.5 (M−H) and 827.5 (M+Na−2H) (negative mode). ¹H NMR (600 MHz,CDC1₃+CD₃OD, 1:1) for the sodium salt: δ=0.89 (t, J=6.5 Hz, 6H, 2CH3),1.27 (br s, 44H, 22 CH2), 1.54 (m, 4H, 2 OCH2CH2), 3.35-3.43 (m, 8H),3.45 (m, 1H), 3.50 (m, 2H), 3.53-3.59 (m, 3H), 3.66-3.71 (m, 2H),3.77-3.83 (m, 4H). ¹H NMR (500 MHz, CDC1₃+CD₃OD, 1:1) for the ammoniumsalt: δ=0.89 (t, J=7.0 Hz, 6H, 2 CH3), 1.27 (br s, 44H, 22 CH2), 1.55(m, 4H, 2 CH2), 3.37-3.45 (m, 12H), 3.57-3.65 (m, 4H), 3.81-3.90 (m, 4H).

Example 21 Preparation of Compound 26

Compound 2 (9.50 g, 22.08 mmol) was dissolved in dry pyridine (57 mL).p-Toluenesulfonyl chloride (TsCl, 7.16 g, 37.54 mmol) was added at 0° C.to the reaction flask. The reaction was warmed to room temperaturenaturally and stirred overnight. Another portion of tosyl chloride(TsCl, 5.46 g) was added and the mixture was stirred at room temperaturefor 20 h. The solution was concentrated in vacuo. The residue wasco-distilled with toluene. The crude product was dissolved in ethylacetate (600 mL) and transferred to a separatory funnel. The organiclayer was washed with saturated sodium bicarbonate solution (300 mL).The aqueous layer was back-washed with ethyl acetate (300 mL) and thecombined organic layer was dried over sodium sulphate (Na SO₄) andconcentrated. The residue was purified by silica gel chromatography(hexane:ethyl acetate, 3:1 and then 2:1) to give 26 (9.18 g, 59%).TLC:R_(f)=0.25 (hexane:ethyl acetate, 3:1). ¹H NMR (400 MHz, CDC1₃): δ2.41 (s, 3H, CH₃), 2.44 (s, 3H, CH₃), 3.27 (s, 2H), 3.70 (s, 2H), 3.73(d, J=12.0 Hz, 2H), 3.80 (d, J=12.0 Hz, 2H), 3.87 (s, 2H), 3.93 (d,J=12.0 Hz, 2H), 4.00 (d, J=12.0 Hz, 2H), 4.32 (s, 2H), 5.30 (s, 2H,CHPh), 5.41 (s, 1H, CHPh), 7.28-7.48 (m, 14H, Ar—H), 7.81 (m, 4H, Ar—H).

Example 22 Preparation of Compound 27

Compound 26 (9.12 g, 12.91 mmol) was dissolved in toluene (150 mL).Saturated sodium bicarbonate (150 mL), phase transfer catalyst ALIQUAT™(1 mL), and sodium azide (33.58 g, 516.47 mmol) were added to thereaction flask. The solution was heated to 110° C. and stirredovernight. More ALIQUAT™ (1 mL) and sodium azide (16.79 g) were addedand the mixture was stirred at 110° C. for another 4 h. The reaction wasincomplete. The solution was cooled to room temperature and usualaqueous work-up afforded syrup which was purified

The filtrate was concentrated in vacuo. The residue was purified bysilica gel chromatography (hexane:ethyl acetate, 5:1 and 4:1) to give 27(2.58 g,) and the mono-azide substituted intermediate (3.73 g). Themono-azide substituted intermediate (3.73 g) was re-reacted in toluene(40 mL) with saturated sodium bicarbonate solution (40 mL), aliquat (1mL), and sodium azide (16.34 g) at 110° C. for 7 days and more 27 (1.81g) was obtained, resulting in the total yield of 71%. TLC:R_(f)=0.63(hexane:ethyl acetate, 3:1). ¹H NMR (400 MHz, CDC1₃) for one isomer: δ3.30 (s, 4H), 3.78 (d, J=12.0 Hz, 4H), 3.90 (s, 4H), 4.10 (d, J=12.0 Hz,0.4H), 5.40 (s, 2H, 2 CHPh), 7.34-7.48 (m, 10H, Ar—H).

Example 23 Preparation of Compound 28

To diazido compound 27 (0.783 g, 1.629 mmol) in methanol (8 ml) weredropwise added 1,3-propane dithiol (3.27 mL, 32.586 mmol) andtriethylamine (4.54 mL, 32.586 mmol) and the mixture was stirredovernight at room temperature. TLC (hexane:ethyl acetate, 2:1) showedreaction was complete. The reaction mixture was rotoevaporated anddithiol was co-evaporated with chloroform (3×20 mL). The residue waspurified by flash chromatography to give 28 (0.322 g, 46% combinedyield). TLC:R_(f)=0.29 (methanol:dichloromethane:water:ammoniumhydroxide, 9:1.5:0.1:0.1). C₂₄H₃₂N₂O₅ (428.23). ES-MS (positive mode,m/z) found:429 (M+H). ¹H NMR (500 MHz, CDCl₃): δ 2.60 (s, 2H), 3.15 (s,2H), 3.35 (s, 2H), 3.70 (d, J=12.0 Hz, 2H), 3.77 (s, 4H), 3.81 (d,J=12.0 Hz, 2H), 4.09 (d, J=12.0 Hz, 4H), 5.40 (s, 2H), 7.32-7.47 (m,10H).

Example 24 Preparation of Compound 29

Compound 28 (0.160 g, 0.376 mmol) was dissolved in dry dichloromethane(10 mL). 1,3-Dicyclohexylcarbodiimide (DCC, 0.465 g, 2.254 mmol) anddi-lipid acid 7 (0.513 g, 1.128 mmol) were added and the mixture wasstirred at room temperature 60 h. TLCs(chloroform:methanol:water:ammonium hydroxide, 9:1.7:0.1:0.1;methanol:dichloromethane, 5:95; hexane:ethyl acetate, 3:1) showedreaction was complete. Excessive DCC was quenched with water (few drops)and stirred for 15 min. The reaction mixture was filtered through 2×NaSO₄ beds and the precipitate washed with dichloromethane. Rotoevaporatedto a crude yellowish syrup. The syrup was purified by silica gelchromatography (hexane:ethyl acetate, 3:1) to give 29 (0.324 g, 66%yield). C₈₀H₁₃₆N₂O₁₁ (1301.01). ES-MS (positive mode, m/z) found: 1302(M+H), 1324 (M+Na). ¹H NMR (600 MHz, CDCl₃): δ 0.86 (t, J=6.5 Hz, 12H, 4CH3), 1.25 (br s, 80H), 1.60 (m, 8H), 2.25 (m, 4H), 2.46 (m, 2H), 2.58(m, 2H), 3.06 (s, 2H), 3.14 (m, 2H), 3.63 (m, 2H), 3.72-3.80 (m, 6H),3.94-4.02 (m, 4H), 5.18 (m, 1H), 5.26 (m, 1H), 5.38 (s, 1H), 5.43 (s,1H), 6.08 (t, J=7.0 Hz, 1H, H), 7.30-7.46 (m, 10H), 7.84 (t, J=6.5 Hz,1H, NH).

Example 25 Preparation of Compound 30

To a solution of 29 (0.300 g, 0.229 mmol) in anhydrous THF (10 mL) wasadded molecular sieves (4 A, 1 g), and sodium cyanoborohydride (0.287 g,4.593 mmol). The mixture was stirred at 0° C. under nitrogen atmospherefor 15 minutes. Dropwise added ether-HCl (sat) until bubbling stopped(1-2 mL). TLC (hexane:ethyl acetate, 6:4) showed some mono-ring opening.Added another 20 eguivalents (0.287 g) of sodium cyanoborohydride anddropwise added ether-HCl (2 mL). The mixture was filtered over celiteand rotoevaporated off THF under high vacuum. The mixture was dissolvedinto saturated sodium bicarbonate solution (75 ml) and extracted withdichloromethane (3×75 ml). The combined organic layers were washed withsaturated sodium chloride solution (50 ml) and dried with sodiumsulfate, and concentrated to yellow syrup. The syrup was purified bysilica gel chromatography (hexane:ethyl acetate, 6:4) to give compound30 (0.212 g, 71%). TLC:R_(f)=0.17 (hexane:ethyl acetate, 6:4).C₈₀H₁₄₀N₂O₁₁ (1304.04). 1H NMR (300 MHz, CDCl3): δ 0.88 (t, J=6.5 Hz,12H, 4 CH3), 1.25 (br s, 80H), 1.60 (m, 8H), 1.94 (br s, 2H), 2.25 (m,4H), 2.43 (m, 4H), 3.08 (m, 3H), 3.22 (m, 2H), 3.33 (m, 5H), 3.40-3.57(m, 4H), 3.68 (m, 1H), 3.78 (m, 1H), 4.43 (d, J=12.0 Hz, 1H), 4.48 (s,2H), 4.51 (d, J=12.0 Hz, 1H), 5.16 (m, 2H), 6.73 (m, 1H), 6.85 (m, 1H,NH), 7.30 (m, 10H).

Example 26 Preparation of Compound 31

To compound 30 (0.237 g, 0.181 mmol) in anhydrous dichloromethane (5 mL)was added tetrazole (0.076 g, 1.089 mmol) and dropwise-added dibenzyldiisopropyl phosphoramidite (0.250 g, 0.24 ml, 0.724 mmol). The mixturewas stirred at room temperature for 1 hour and the formation of complexchecked with TLC (hexane:ethyl acetate, 6:4). Added another 3equivalents (0.038 g) of tetrazole and 2 equivalents (0.125 mg) ofphosphoramidite. TLC after 2 hours showed the starting material wascompletely consumed. The mixture was then cooled to 0° C. and3-chloroperbenzoic acid (0.312 g, 1.81 mmol) was added slowly resultingin gas formation. After 30 min, the mixture was poured into 10% sodiumhydrogen sulfite (40 ml) and extracted with dichloromethane (3×40 ml).The organic layer was washed with saturated sodium bicarbonate solution(20 ml), dried with sodium sulfate and concentrated to a yellow syrup.The syrup was purified by repeatedchromatography (hexane:ethyl acetate,7:3; hexane:acetone, 4:1) to give 31 (198 mg, 60%). TLC:Rf=0.28(hexane:ethyl acetate, 3:1). C₁₀₈H₁₆₆N₂O₁₇P₂ (1825.16). ES-MS (negativemode, m/z) found: 1861 (M+Cl). 1H NMR (500 MHz, CDC1₃): δ 0.88 (t, J=6.5Hz, 12H, 4 CH₃), 1.25 (br s, 80H), 1.55 (m, 8H), 2.21 (2 t, J=6.5 Hz,each 2H), 2.39 (m, 4H), 3.10 (m, 2 H), 3.16 (m, 4H), 3.31 (m, 6H), 3.95(m, 4H), 4.37 (m, 4H), 4.98 (m, 8H), 5.19 (m, 2H), 7.08 (t, J=6.0 Hz,2H, 2 NH), 7.22-7.31 (m, 30H).

Example 27 Preparation of Compound 4

To a solution of 31 (28 mg, 0.015 mmol) in distilled THF-AcOH (10:1, 75mL) was added palladium on carbon (10%, 75 mg). The mixture was stirredat room temperature under hydrogen atmosphere for 16 hrs. TLC(chloroform:methanol:ammonium hydroxide:water, 2:3:0.5:0.5) indicatedmostly product. The solid was gravity filtered and the filtrateconcentrated under high vacuum. The residue was purified by flashchromatography using Iatrobeads as support (chloroform:methanol, 9:1 tochloroform:methanol:water, 5:3:0.3) to give 4 (18 mg, 95% yield).TLC:R_(f)=0.20 (chloroform:methanol:water, 4:2:0.3). C₆₆H₁₃₀N₂O₁₇P₂(1284.88). ES+MS (negative mode, m/z) found: 1284 (M−H). ¹H NMR (600MHz, CDCl₃+CD₃OD, 1:1): δ 0.85 (t, J=6.5 Hz, 12H, 4 CH₃), 1.28 (br s,80H), 1.62 (m, 8H), 2.30 (m, 4H), 2.50-2.63 (m, 4H), 3.03-3.15 (m, 4H),3.20-3.31 (m, 6H), 3.34-3.38 (m, 2H), 3.70-3.78 (m, 4H), 5.28 (m, 2H).

Example 28

Induction of Cytokine Secretion by Lipid A Analogs Adherent cellsisolated from human peripheral blood were incubated in completeRPMI-1640 medium in the presence of GM-CSF and IL-4. After three days ofincubation, the lipid A analogs were added at a concentration of 10pg/mL. After 24 hour of incubation, the supernatants were harvested andthe presence of the cytokines was determined using ELISA kits.TNF-alpha, IL-6 and IL-8 levels were measured and listed in Table 1(also see FIG. 14).

TABLE 1 In vitro cytokine secretion pattern of human adherent cellsactivated with synthetic lipid A mimic 1, 2, or R595 lipid A*. TNF-alpha(pg/mL) IL-6 (pg/mL) IL-8 (pg/mL) 1 9723 21016 97980 2 4591 14097 72868R595 5490 19424 82612 lipid A Medium 263 17 99 *R595 lipid A is thenatural lipid A product isolated from Salmonella minnesota, R595 (AvantiPolar Lipids, Inc.)

Example 29 Mice Immunized with Liposomal Vaccines

Groups of C57-Black mice were immunized subcutaneously with the BLP25liposomal vaccine containing 0.2-200 pg of MUCl-based 25-mer lipopeptideas an antigen, which has the peptide sequence ofH₂N-STAPPAHGVTSAPDTRPAPGSTAPPK (Pal) G-OH, and 0.1-100 μg (half weightof the lipopeptide antigen) of lipid A analog per dose. Nine days aftervaccine injection, mice were sacrificed and lymphocytes were taken fromthe draining lymph nodes (local response) or from the spleens (systemicresponse) to determine the immune response in each group. Thelymphocytes taken from immunized mice were incubated in in vitrocultures in the presence of MUCl-based boosting antigen BP1-151, whichhas the peptide sequence H₂N-STAPPAHGVTSAPDTRPAPGSTAPPK-OH.

Example 30 Measurement of T-cell Proliferation

T-cell proliferation was evaluated using a standard 3H thymidineincorporation assay. Briefly, nylon wool passed inguinal lymph nodelymphocytes from each mouse were added to a culture containing 106native mitomycin C treated syngeneic splenocytes, which serves asantigen presenting cells (APCs). To each well 20 μg of MUC1-basedboosting peptide BP1-151, H₂N-STAPPAHGVTSAPDTRPAPGSTAPPK-OH, was addedfor positive control; and cultures containing no antigen or peptideBP1-72, which has the peptide sequence H2N-EAIQPGCIGGPKGLPGLPGP-OH, wereused as negative control. The culture was incubated for 72 h in a totalvolume of 250 μl/well, followed by adding 1 μCi of ³H-thymidine in avolume of 50 μl. The plates were incubated for an additional 18-20 h.Cells were harvested and [³H]dTh incorporation was measured by liquidscintillation counter. T-cell proliferation results corresponding tovarious liposomal vaccines adjuvanted with lipid A mimic 1, 2, or R595lipid A are shown in Table 2 and FIG. 15.

TABLE 2 Antigen specific T cell proliferation response afterimmunization of C57BL/6 mice with one dose of BLP25 liposomal vaccine.The dose contains 0.2 lag of 25-mer MUC1 based lipopeptide antigen and0.1 pg of lipid A analog (1, 2, and R595 lipid A) as the adjuvant.Compound CPM (counts per minute) SD 1 14831 ±2475 2 20793 ±2505 R595lipid A 11920 ±3630 Saline 320 ±292

Example 31 Inhibition of Tumor Growth by Liposomal vaccine adjuvanted bySynthetic Lipid A Mimics

C57BL/6 mice were challenged subcutaneously with MC38-MUC1 tumor cellson day 0. On day 7, 14, and 21, the groups of mice were immunizedintradermally with BLP25 liposomal vaccine containing 200 μg/dose ofMUC1 based 25-mer lipopeptide antigen and 100 μg/dose of synthetic lipidA mimic 1, 2, or R595 lipid A. On day 34, the tumor diameters (lengthand width) were taken with a caliper and tumor size ware expressed inmm² (length×width). The data is shown in Table 3 and FIG. 16.

TABLE 3 Active specific immunotherapy of MC-38 MUC1 tumor bearing miceimmunized intradermally with BLP25 liposomal vaccine containing lipid Aanalogs (1, 2, and R595 lipid A). The vaccine dose contains 200 μg ofBLP25 lipopeptide and 100 μg of lipid A analog as adjuvant. CompoundTumor size (mm²) 1 284 2 298 R595 lipid A 287 Saline 539

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-   M. P. Fink, Crit. Care Med. 1993, 21 Suppl.: S32-S39.-   W. J. Christ, T. Kawata, L. D. Hawkins, S, Kobayashi, O. Asano,    and D. P. Rossignol. European patent EP-536969-A2. Dement    Publications. Ltd.-   Citation of documents herein is not intended as an admission that    any of the documents cited herein is pertinent prior art, or an    admission that the cited documents is considered material to the    patentability of any of the claims of the present application. All    statements as to the date or representation as to the contents of    these documents is based on the information available to the    applicant and does not constitute any admission as to the    correctness of the dates or contents of these documents.

The appended claims are to be treated as a non-limiting recitation ofpreferred embodiments.

In addition to those set forth elsewhere, the following references arehereby incorporated by reference, in their most recent editions as ofthe time of filing of this application: Kay, Phage Display of Peptidesand Proteins: A Laboratory Manual; the John Wiley and Sons CurrentProtocols series, including Ausubel, Current Protocols in MolecularBiology; Coligan, Current Protocols in Protein Science; Coligan, CurrentProtocols in Immunology; Current Protocols in Human Genetics; CurrentProtocols in Cytometry; Current Protocols in Pharmacology; CurrentProtocols in Neuroscience; Current Protocols in Cell Biology; CurrentProtocols in Toxicology; Current Protocols in Field AnalyticalChemistry; Current Protocols in Nucleic Acid Chemistry; and CurrentProtocols in Human Genetics; and the following Cold Spring HarborLaboratory publications: Sambrook, Molecular Cloning: A LaboratoryManual; Harlow, Antibodies: A Laboratory Manual; Manipulating the MouseEmbryo: A Laboratory Manual; Methods in Yeast Genetics: A Cold SpringHarbor Laboratory Course Manual; Drosophila Protocols; Imaging Neurons:A Laboratory Manual; Early Development of Xenopus laevis: A LaboratoryManual; Using Antibodies: A Laboratory Manual; At the Bench: ALaboratory Navigator; Cells: A Laboratory Manual;

Methods in Yeast Genetics: A Laboratory Course Manual; DiscoveringNeurons The Experimental Basis of Neuroscience; Genome Analysis: ALaboratory Manual Series; Laboratory DNA Science; Strategies for ProteinPurification and Characterization: A Laboratory Course Manual; GeneticAnalysis of Pathogenic Bacteria: A Laboratory Manual; PCR Primer: ALaboratory Manual; Methods in Plant Molecular Biology: A LaboratoryCourse Manual; Manipulating the Mouse Embryo: A Laboratory Manual;Molecular Probes of the Nervous System; Experiments with Fission Yeast:A Laboratory Course Manual; A Short Course in Bacterial Genetics: ALaboratory Manual and Handbook for Escherichia coli and RelatedBacteria; DNA Science: A First Course in Recombinant DNA Technology;Methods in Yeast Genetics: A Laboratory Course Manual; Molecular Biologyof Plants: A Laboratory Course Manual.

All references cited herein, including journal articles or abstracts,published, corresponding, prior or otherwise related U.S. or foreignpatent applications, issued U.S. or foreign patents, or any otherreferences, are entirely incorporated by reference herein, including alldata, tables, figures, and text presented in the cited references.Additionally, the entire contents of the references cited within thereferences cited herein are also entirely incorporated by reference.

Reference to known method steps, conventional methods steps, knownmethods or conventional methods is not in any way an admission that anyaspect, description or embodiment of the present invention is disclosed,taught or suggested in the relevant art.

The foregoing description of the specific embodiments will so fullyreveal the general nature of the invention that others can, by applyingknowledge within the skill of the art (including the contents of thereferences cited herein), readily modify and/or adapt for variousapplications such specific embodiments, without undue experimentation,without departing from the general concept of the present invention.Therefore, such adaptations and modifications are intended to be withinthe meaning and range of equivalents of the disclosed embodiments, basedon the teaching and guidance presented herein. It is to be understoodthat the phraseology or terminology herein is for the purpose ofdescription and not of limitation, such that the terminology orphraseology of the present specification is to be interpreted by theskilled artisan in light of the teachings and guidance presented herein,in combination with the knowledge of one of ordinary skill in the art.

Any description of a class or range as being useful or preferred in thepractice of the invention shall be deemed a description of any subclass(e.g., a disclosed class with one or more disclosed members omitted) orsubrange contained therein, as well as a separate description of eachindividual member or value in said class or range.

The description of a minimum and the separate description of a maximum,where the maximum is greater than the minimum, imply that in a preferredembodiment the two may be combined to form a fully close-ended range. Ifthe maximum equals the minimum, a preferred value is implied.

The description of preferred embodiments individually shall be deemed adescription of any possible combination of such preferred embodiments,except for combinations which are impossible (e.g., mutually exclusivechoices for an element of the invention) or which are expressly excludedby this specification.

The term “comprising”, as used in the claims herein, means that theelements subsequently recited are required, but that the inclusion ofadditional elements is allowed if not expressly excluded by some otherlimitation.

The word “a”, unless otherwise qualified, implies “one or more”.

If an embodiment of this invention is disclosed in the prior art, thedescription of the invention shall be deemed to include the invention asherein disclosed with such embodiment excised.

The invention, as contemplated by applicant (s), includes but is notlimited to the subject matter set forth in the appended claims, andpresently unclaimed combinations-thereof. It further includes suchsubject matter further limited, if not already such, to that whichovercomes one or more of the disclosed deficiencies in the prior art. Tothe extent that any claims encroach on subject matter disclosed orsuggested by the prior art, applicant (s) contemplate the invention (s)corresponding to such claims with the encroaching subject matterexcised.

All references, including patents, patent applications, books, articles,and online sources, cited anywhere in this specification are herebyincorporated by reference, as are any references cited by saidreferences.

1-183. (canceled)
 184. A method of stimulating the immune system of asubject comprising administering to a subject having cancer (i) animmunogen and; (ii) an immunostimulatory amount of a compound having thestructure (I)

wherein at least one of R₁, R₂, R₃, R₅, R₆ and R₇ is a stronglylipophilic group selected from the group consisting of

wherein X, X₁, X₂, and X₃ are independently —CO— or —CH₂; Z is —NH— or—O—; k, m, and r are independently an integer of 0 to 30 inclusive, nand q are independently an integer of 0 to 6 inclusive; wherein Y4 is aspacer selected from the group consisting of —O—, —S—, and —NH—,wherein, at least one of Y₁R₁, Y₂R₂, Y₃R₃, Y₅R₅, Y₆R₆ and Y₇R₇ is amonovalent phosphate equivalent (MPE), wherein each monovalent phosphateequivalent is, independently, (a) —R′—C(O)OH, where R′ is a substitutedor unsubstituted alkyl group of 1-4 carbons, or (b) selectedindependently from the group consisting of —OB(OH)OR, —OP(O)(OH)OR,—PS(O)(O)(OH)OR, and —OP(═O)(OH)—O—P(═O)(OH)OR, where R is hydrogen, ora substituted or unsubstituted alkyl group of 1-4 carbons, and if R is asubstituted alkyl group, the substitutions are —OH or —NH₂. wherein R₈is selected from the group consisting of H, OH, OR₉, a moiety which incombination with Y₈ forms a monovalent phosphate equivalent aspreviously defined, and a group (i)-(viii) as defined above; wherein R9is an alkyl or acyl group of 1 to 10 carbon length; and wherein theglycosidic linkage is α or β; or an immunostimulatory amount of acompound having the structure (II)

wherein at least one of R₁, R₂, R₃, R₁₁, R₁₂ and R₁₃ is a stronglylipophilic group selected from the group consisting of (i)-(viii) above;wherein Y4 is a spacer selected from the group consisting of —O—, —S—,and —NH—, and wherein, at least one of Y₁R₁, Y₂R₂, Y₃R₃, Y₁₁R₁₁, Y₁₂R₁₂and Y₁₃R₁₃ is independently a monovalent phosphate equivalent aspreviously defined; wherein the following limitations apply to both (I)and (II) above: Y₁, Y₂, Y₃, Y₅, Y₆, Y₇, Y₁₁, Y₁₂ and Y₁₃ are spacersindependently selected from the group consisting of —O—, —S—, and —NH—;R₁, R₂, R₃, R₅, R₆, R₇, R₁₁, R₁₂ and R₁₃ are independently hydrogen, amoiety which with the commonly numbered Y group forms monovalentphosphate equivalent as previously defined, or a strongly lipophilicgroup selected from the group consisting of (i)-(viii) above, thestrongly lipophilic groups of said compound collectively provide atleast two major carbon chains, and the major carbon chains of saidstrongly lipophilic groups collectively provide at least 30 carbonatoms; or which compound is a pharmaceutically acceptable salt of (I) or(II).
 185. The method of claim 184, wherein Y4 is —O—.
 186. The methodof claim 184, wherein Y₁, Y₂, Y₃, Y₄, Y₅, Y₆, Y₇, Y₁₁, Y₁₂, and Y₁₃ areindependently —O— or —NH—.
 187. The method of claim 184, wherein eachmonophosphate equivalent is —OP(O)(OH)(OH).
 188. The method of claim184, wherein Y₄ is —O—; Y₁, Y₂, and Y₇ are —O—; Y₃, Y₅ and Y₆ areindependently —O— or —NH—; R₁, R₃, R₅ and R₆ are independently hydrogenor a strongly lipophilic group selected from (i)-(viii); at least one ofR₁, R₃, R₅ and R₆ is not hydrogen; R₂ and R₇ are independently selectedfrom the group consisting of H, —P(O)(OH)₂, —SO₃H,—P(O)(OH)(OCH₂CH₂NH₂), and —CH₂COOH; and R₈ is selected from the groupconsisting of H, OH, OSO₃H, and OR₉ wherein R₉ is an alkyl or acyl groupof 1 to 10 carbon length.
 189. The method of claim 184, wherein R₁, R₃,R₅ and R₆ are independently hydrogen or a strongly lipophilic groupselected from the group consisting of (i)-(viii), at least one R₁, R₃,R₅ and R₆ is not hydrogen, and R₂ and R₇ are independently selected fromthe group consisting of H, —P(O)(OH)₂, —SO₃H, —P(O)(OH)(OCH₂CH₂NH₂), and—CH₂COOH; and R₈ is selected from the group consisting of H, OH, OSO₃H,and OR₉ wherein R₉ is an alkyl or acyl group of 1 to 10 carbon length.190. The method of claim 184, wherein R₁, R₃, R₁₁, and R₁₃ areindependently hydrogen, or a strongly lipophilic group selected from(i)-(viii); at least one of R₁, R₃, R₅ and R₆ is not hydrogen; and R₂and R₁₂ arc independently selected from the group consisting of H,—P(O)(OH)₂, —SO₃H, —P(O)(OH)(OCH₂CH₂NH₂), and —CH₂COOH.
 191. The methodof claim 184, wherein at least one strongly lipophilic group is one ofthe structures set forth below


192. The method of claim 188, wherein the structure is further definedas the following

wherein R₁, R₃, R₅ and R₆ are independently hydrogen or a lipophilicgroup selected from the group consisting of

wherein X, X₁, X₂, and X₃ are independently —CO— or —CH₂; Z is —NH— or—O—; k, m, and r are independently an integer of 0 to 30 inclusive, nand q are independently an integer of 0 to 6 inclusive; at least one ofR₁, R₃, R₅ and R₆ is not hydrogen; R₂ and R₇ are independently selectedfrom the group consisting of H, —P(O)(OH)₂, —SO₃H,—P(O)(OH)(OCH₂CH₂NH₂), and —CH₂COOH; and R₉ is H, or an alkyl or acylgroup of 1 to 10 carbon length.
 193. The method of claim 192, wherein R₁and R₉ are hydrogen; R₂ is a hydrogen or the phosphono group —P(O)(OH)₂;R₇ is the phosphono group —P(O)(OH)₂; and R₃, R₅ and R₆ are the same ordifferent acyl groups of the following structure

wherein m and n are independently chosen from an integer between 6 to 10inclusive.
 194. The method of claim 193, having the following structure


195. The method of claim 193, having the following structure


196. The method of claim 184, wherein the immunogen comprises a peptideepitope.
 197. The method of claim 184, wherein the immunogen comprises aMUC1 epitope.
 198. The method of claim 184, wherein the immunogencomprises a strongly lipophilic group.
 199. The method of claim 184,wherein the immunogen, immunostimulatory compound, or both is deliveredby means of a liposomal formulation.
 200. (canceled)
 201. (canceled)202. A method for treating cancer in a subject comprising administeringto a subject expressing a cancer associated mucin, (i) an immunogencomprising a MUC1 epitope and; (ii) an immunostimulatory amount of acompound having the structure (I)

wherein at least one of R₁, R₂, R₃, R₅, R₆ and R₇ is a stronglylipophilic group selected from the group consisting of

wherein X, X₁, X₂, and X₃ are independently —CO— or —CH₂; Z is —NH— or—O—; k, m, and r are independently an integer of 0 to 30 inclusive, nand q are independently an integer of 0 to 6 inclusive; wherein Y4 is aspacer selected from the group consisting of —O—, —S—, and —NH—,wherein, at least one of Y₁R₁, Y₂R₂, Y₃R₃, Y₅R₅, Y₆R₆ and Y₇R₇ is amonovalent phosphate equivalent (MPE), wherein each monovalent phosphateequivalent is, independently, (a) —R′—C(O)OH, where R′ is a substitutedor unsubstituted alkyl group of 1-4 carbons, or (b) selectedindependently from the group consisting of —OB(OH)OR, —OP(O)(OH)OR,—OS(O)(O)(OH)OR, and —OP(═O)(OH)—O—P(═O)(OH)OR, where R is hydrogen, ora substituted or unsubstituted alkyl group of 1-4 carbons, and if R is asubstituted alkyl group, the substitutions are —OH or —NH₂, wherein R₈is selected from the group consisting of H, OH, OR₉, a moiety which incombination with Y₈ forms a monovalent phosphate equivalent aspreviously defined, and a group (i)-(viii) as defined above; wherein R₉is an alkyl or acyl group of 1 to 10 carbon length; and wherein theglycosidic linkage is α or β; or an immunostimulatory amount of acompound having the structure (II)

wherein at least one of R₁, R₂, R₃, R₁₁, R₁₂ and R₁₃ is a stronglylipophilic group selected from the group consisting of (i)-(viii) above;wherein Y4 is a spacer selected from the group consisting of —O—, —S—and —NH—, and wherein, at least one of Y₁R₁, Y₂R₂, Y₃R₃, Y₁₁R₁₁, Y₁₂R₁₂and Y₁₃R₁₃ is independently a monovalent phosphate equivalent aspreviously defined; wherein the following limitations apply to both (T)and (II) above: Y₁, Y₂, Y₃, Y₅, Y₆, Y_(7.) Y₁₁, Y₁₂ and Y₁₃ are spacersindependently selected from the group consisting of —O—, —S—, and —NH—;R₁, R₂, R₃, R₅, R₆ R₇. R₁₁, R₁₂ and R₁₃ are independently hydrogen, amoiety which with the commonly numbered Y group forms monovalentphosphate equivalent as previously defined, or a strongly lipophilicgroup selected from the group consisting of (i)-(viii) above, thestrongly lipophilic groups of said compound collectively provide atleast two major carbon chains, and the major carbon chains of saidstrongly lipophilic groups collectively provide at least 30 carbonatoms; or which compound is a pharmaceutically acceptable salt of (I) or(II).
 203. The method of claim 202, wherein the immunogen furthercomprises a peptide epitope.
 204. The method of claim 202, wherein theimmunogen further comprises a strongly lipophilic group.
 205. The methodof claim 202, wherein the immunogen, immunostimulatory compound, or bothis delivered by means of a liposomal formulation.
 206. (canceled) 207.(canceled)